Selection methods for genetically-modified t cells

ABSTRACT

In some aspects, isolated transgenic cells (e.g., transgenic T cells) are provided that comprise or express a transgene and DHFRFS and/or TYMSSS. Methods for selecting transgeneic cells are also provided.

This application is a division of U.S. application Ser. No. 15/552,821,filed Aug. 23, 2017, as a national phase application under 35 U.S.C. §371 of International Application No. PCT/US2016/019288, filed Feb. 24,2016, which claims the benefit of United States Provisional PatentApplication Nos. 62/120,329, filed Feb. 24, 2015, 62/120,790, filed Feb.25, 2015, and 62/175,794, filed Jun. 15, 2015, the entirety of each ofwhich is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSCP1272USD1_ST25.txt”, which is 13 KB (as measured in MicrosoftWindows®) and was created on Sep. 2, 2020, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The disclosure relates to methods and compositions for preparingtransgenic T cells and enriching for regulatory T cells in a populationof T cells isolated from a mammal.

2. Description of Related Art

Targeting T cells to human disease has been in progress for more than 25years. See Yee C., Immunological reviews 2014, 257(1):250-263. Theinitial aim of clinical trials was to direct T cells to target and killdiffuse cancers, for example metastatic melanoma and leukemia. See YeeC., Immunological reviews 2014, 257(1):250-263 and Roddie C and Peggs KS, Expert opinion on biological therapy 2011, 11(4):473-487.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Antigens on cancers are often times overexpressed or mutated versions ofproteins found on non-cancerous cells. Although cancer antigens ideallydemarcate only the cancer, in many instances cancer antigens are foundon non-cancerous cells with the risk of off-tumor toxicities that causeserious complications that many times have led to morbidity and death.The powerful nature of T cell therapies is one of the reasons that Tcells continue to be sought as a therapeutic, but have not yet reachedFDA approval in the United States for any form of disease.

While many of the T cell clinical trials are showing strong benefit overstandard of care, the cost of producing a T cell therapy and risk to thepatient continues to hamper development of these technologies beyond afew specialized centers. Further limitations exist due to the compleximmunosuppressive environment of the tumor, and difficulty ofidentifying appropriate tumor antigens. See Corrigan-Curay J, Kiem H Pet al., Molecular therapy: the Journal of the American Society of GeneTherapy 2014, 22(9):1564-1574. It should be noted that T celltherapeutics in cancer were initially developed for the treatment ofmelanoma and leukemia, and in the intervening quarter century have notsignificantly deviated from those cancer targets. Further improvementsin the technical aspects of T cell therapy as well as continuingresearch and development of immune-modulatory drugs will continue topromote T cell cancer therapies for cancer and potentially broaden theapplicability of these therapeutics.

Diseases of excessive inflammation are currently targeted byimmune-modulatory or immune-suppressive medications. These therapies areoften effective, but have untoward side effects as discussed in theabove section. Better targeted immunosuppression may be possible usingregulatory T cells (T_(regs)). As T_(regs) are better understood andculturing techniques become more advanced, cell therapies based onreconstituting T_(regs) will likely move toward clinical trials morerapidly. The use of T_(regs) in clinical trials has been limited topreventing GvHD following hematopoetic stem cell transplantation (HSCT)for the most part. It is likely that the number of uses for T_(reg) willexpand as many other forms of inflammation have been targeted inpreclinical models. Technical challenges related to the isolation andpropagation of T_(reg) is currently limiting the advance of this T celltherapy. See Singer B D et al., Frontiers in immunology 2014, 5:46.

The development of MEC independent T cell propagation methods has been agreat technical advance for T cell therapies. Growing T cells byantigen-specificity-independent selection (ASIS) generates large numbersof T cells for reinfusion to a patient. While it might seemcounterintuitive to grow T cells without direct selection forspecificity, the large number of T cells can include an activated andpropagated subset of T cells that are specific to the antigen targeted.Novel ASIS methods are sought to enhance the selection of transgenic Tcells and to select for therapeutically useful T cell phenotypes. Whilein vitro ASIS using chimeric cytokine receptors is a recently reportedmethod of non-immunogenic selection, it only utilizes the third signalin T cell activation—cytokine signaling. See Wilkie S et al., TheJournal of biological chemistry 2010, 285(33):25538-25544. A strategythat can utilize the first and second signals of T cell activation (CD3and costimulatory signaling) of human genes to activate and propagate Tcells independent of antigen specificity can be of further benefit.

The adoptive transfer of antigen-specific T cells is a rapidlydeveloping field of cancer immunotherapy with various approaches totheir manufacture being tested and new antigens being targeted. T cellscan be genetically-modified for immunotherapy to express chimericantigen receptors (CAR) that recognize tumor-associated antigens (TAAs)independent of HLA (HLA is the human version of MEC) expression. Recentresults from early-phase clinical trials demonstrate that CAR⁺ T-cell(CART) therapies can lead to partial and complete remissions ofmalignant diseases, including in some recipients with advanced/relapsedB-cell tumors. See Kalos M et al., Science translational medicine 2011,3(95):95ra73 and Kochenderfer J N et al., Blood 2012, 119(12):2709-2720.

Therefore, notwithstanding what has previously been reported in theliterature, there exists a need for improved methods of preparingtransgenic T cells, propagating T cells for therapeutic treatments andselecting for regulatory T cells. Additionally, methods of making andusing transgenic T cells and agents regulating the propagation andselection of transgenic T cells will greatly aid in the treatment ofcancer, autoimmune diseases, infectious diseases and any number of othermedical conditions in which the immune system plays a role.

SUMMARY OF THE INVENTION

In one aspect, an isolated transgenic mammalian T cell comprising orexpressing a transgene and one or more of DHFR^(FS) and TYMS^(SS) isprovided. In some embodiments, the isolated transgenic mammalian T cellcomprises or expresses a transgene, DHFR^(FS) and TYMS^(SS). In someembodiments, the transgene is a suicide gene. In some embodiments, asuicide gene is further included. In some embodiments, codonoptimization is performed on DHFR^(FS), TYMS^(SS), or both.

In another aspect is provided a method for inhibiting anti-thymidylate(AThy) toxicity in a mammalian T cell comprising expressing ananti-thymidylate resistance (AThyR) transgene in said mammalian T cell.In some embodiments, the AThyR transgene is DHFR^(FS). In someembodiments, the AThyR transgene is TYMS^(SS). In some embodiments, thetransgene is a suicide gene. In some embodiments, a suicide gene isfurther included. In some embodiments, codon optimization is performedon DHFR^(FS), TYMS^(SS), or both.

In another aspect is provided a method for selecting a T cell expressinga transgene of interest. The method comprises applying a thymidinesynthesis inhibitor to a plurality of T cells that comprises a T cellexpressing the transgene of interest and DHFR^(FS) and selecting for oneor more T cells surviving after seven or more days of application of thethymidine synthesis inhibitor, wherein the one or more T cells expressesa vector comprising the transgene of interest and DHFR^(FS). Thethymidine synthesis inhibitor may be selected from the group consistingof methotrexate (MTX), 5-FU, Raltitrexed and Pemetrexed. In someembodiments, the transgene is a suicide gene. In some embodiments, asuicide gene is further included. In some embodiments, codonoptimization is performed on DHFR^(FS), TYMS^(SS), or both.

Yet another aspect is a method for selectively propagating peripheralblood mononuclear cells (PBMC) resistant to MTX and 5-FU. The methodcomprises transfecting peripheral PBMC with a vector comprising an AThyRgene, treating the transfected PBMC with a thymidine synthesis inhibitorand selecting for PBMC that express the AThyR gene. In some embodimentsof this aspect, the method further comprises propagating a T cellpopulation from the transfected PBMC. In some embodiments, the thymidinesynthesis inhibitor may be selected from the group consisting ofmethotrexate (MTX), 5-FU, Raltitrexed and Pemetrexed. In someembodiments, the thymidine synthesis inhibitor is MTX. In someembodiments, the AThyR gene is TYMS^(SS). In some embodiments, the AThyRgene is DHFR^(FS). In some embodiments, codon optimization is performedon DHFR^(FS), TYMS^(SS), or both.

Another aspect is an isolated transgenic mammalian T cell comprising anucleic acid sequence comprising a transgene of interest and anucleotide sequence encoding DHFR^(FS) or TYMS^(SS). In someembodiments, the isolated transgenic mammalian T cell comprises anucleic acid comprising a transgene of interest and a nucleotidesequence encoding DHFR^(FS), wherein the transgene of interest and thenucleotide sequence encoding DHFR^(FS) are operably linked. In someembodiments, the isolated transgenic mammalian T cell comprises anucleic acid comprising a transgene of interest and a nucleotidesequence encoding TYMS^(SS), wherein the transgene of interest and thenucleotide sequence encoding TYMS^(SS) are operably linked. In someembodiments, the transgene is a suicide gene. In some embodiments, asuicide gene is further included. In some embodiments, codonoptimization is performed on DHFR^(FS), TYMS^(SS), or both.

In another aspect is provided an isolated transgenic mammalian T cellexpressing a transgene and DHFR^(FS), wherein the T cell comprises (1) apolynucleotide comprising sequence that encodes the transgene and (2) apolynucleotide comprising sequence that encodes the DHFR^(FS). In someembodiments, the transgene is a suicide gene. In some embodiments, asuicide gene is further included. In some embodiments, codonoptimization is performed on DHFR^(FS).

In another aspect is provided an isolated transgenic mammalian T cellexpressing a transgene and TYMS^(SS), wherein said T cell comprises (1)a polynucleotide comprising sequence that encodes the transgene and (2)a polynucleotide comprising sequence that encodes the TYMS^(SS). In someembodiments, the transgene is a suicide gene. In some embodiments, asuicide gene is further included. In some embodiments, codonoptimization is performed on TYMS^(SS).

In yet another aspect is provided a method of treating a patient with acancer comprising administering to a patient a therapeutically effectiveamount of a T cell of an isolated T cell of any of the aboveembodiments.

In some embodiments, a combination therapy of AThyR⁺ T cells with AThytherapies can be used to improve anti-tumor immunity. An isolated T cellwith a AThyR⁺ phenotype can be administered with MTX, 5-FU, Raltitrexedand Pemetrexed, or any other thymidine synthesis inhibitor.

In yet another aspect is provided a method for selecting for a T cellexpressing a transgene of interest, as shown in any of the FIGS. or asdescribed in the description.

In yet another aspect is provided a T cell, as shown in any of the FIGS.or as described in the description.

In another aspect is a method for selectively propagating human T cellsresistant to one or more of MTX, 5-FU, Raltitrexed and Pemetrexed, asshown in any of the FIGS. or as described in the description. In someembodiments, the human T cells are primary human T cells.

Another aspect is a method of enriching for regulatory T cells in apopulation of T cells isolated from a mammal by contacting saidpopulation with a thymidine synthesis inhibitor selected from the groupconsisting of MTX, 5-FU, Raltitrexed and Pemetrexed, or a combinationthereof, to selectively deplete effector T cells in the population. Insome embodiments, the population of T cells isolated from a mammal iscontacted with both MTX and 5-FU. In some embodiments, the T cellsexpress one or more of DHFR^(FS) and TYMS^(SS). In some embodiments, theT cells express both DHFR^(FS) and TYMS^(SS). In some embodiments, codonoptimization is performed on DHFR^(FS), TYMS^(SS), or both.

Another aspect is a method for depleting regulatory T cells in apopulation of T cells isolated from a mammal by culturing saidpopulation in the presence of one or more aminoglycosidases toselectively deplete the regulatory T cells in said culture. In someembodiments, the T cells express one or more of DHFR^(FS) and TYMS^(SS).In some embodiments, the T cells express both DHFR^(FS) and TYMS^(SS).In some embodiments, codon optimization is performed on DHFR^(FS),TYMS^(SS), or both.

Another aspect is a method for selecting for a regulatory T cellisolated from a mammal. The method comprises treating a plurality of Tcells expressing one or more of DHFR^(FS) and TYMS^(SS) with a thymidinesynthesis inhibitor and selecting a regulatory T cell that expresses amarker for a regulatory T cell. In some embodiments, the T cells expressDHFR^(FS). In some embodiments, the selecting step comprises cellisolating with magnetic bead sorting using one or more of an anti-CD4antibody, an anti-CD25 antibody, an anti-CD3 antibody, an anti-CD8antibody, an anti-CD25 antibody, an anti-CD39 antibody, an anti-CD45antibody, an anti-CD152 antibody, an anti-KI-67 antibody, an anti-LAPantibody and an anti-FoxP3 antibody. In some embodiments, the thymidinesynthesis inhibitor is selected from the group consisting ofmethotrexate (MTX), 5-FU, Raltitrexed or Pemetrexed. In someembodiments, the method further comprises treating the regulatory T cellwith one or more of folate, leucovarin and FU.

In another aspect is provided a composition comprising a first pluralityof T cells isolated from a mammal and a thymidine synthesis inhibitor.The first plurality of T cells is enriched for regulatory T cells ascompared to a second plurality of T cells isolated from a mammal thatdoes not comprise a thymidine synthesis inhibitor.

With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

In another aspect is provided an isolated transgenic mammalian T cellexpressing a transgene and DHFR^(FS), wherein the T cell comprises (1) apolynucleotide comprising sequence that encodes the transgene and (2) apolynucleotide comprising sequence that encodes the DHFR^(FS). In someembodiments, codon optimization is performed on DHFR^(FS) and/or thesequence encoding the transgene of interest. In some embodiments, thetransgene of interest and the nucleotide sequence encoding DHFR^(FS),upon expression, are encoded on the same mRNA. In further embodiments,the sequence encoding the transgene of interest and the nucleotidesequence encoding DHFR^(FS) are separated by an internal ribosomal entrysite (IRES) or a ribosomal slip sequence. In certain embodiments, thetransgene of interest may encode a chimeric antigen receptor (CAR)construct, a T-cell Receptor (TCR), a hormone (e.g., glucagon), acytokine, a chemokine, a suicide gene, a transcription factor or a cellsurface polypeptide, such as a receptor (e.g., an integrin, cytokinereceptor, chemokine receptor or hormone receptor).

In another aspect is provided an isolated transgenic mammalian T cellexpressing a transgene and TYMS^(SS), wherein said T cell comprises (1)a polynucleotide comprising sequence that encodes the transgene and (2)a polynucleotide comprising sequence that encodes the TYMS^(SS). In someembodiments, codon optimization is performed on TYMS^(SS) and/or thesequence encoding the transgene of interest. In certain embodiments, thetransgene of interest and the nucleotide sequence encoding TYMS^(SS),upon expression, are encoded on the same mRNA. In some embodiments, thesequence encoding the transgene of interest and nucleotide sequenceencoding TYMS^(SS) are separated by an IRES or a ribosomal slipsequence. In specific embodiments, the isolated transgenic mammalian Tcell expressing a transgene and TYMS^(SS) further comprises a nucleotidesequence encoding DHFR^(FS) (optionally, the nucleotide sequenceencoding DHFR^(FS) is operably linked to a second transgene ofinterest). In some embodiments, the transgene of interest (e.g.,operably linked to TYMS^(SS)) is a growth factor, a CAR construct, aTCR, a hormone (e.g., glucagon), a cytokine, a chemokine, a suicidegene, a transcription factor (e.g., FoxP3) or a cell surfacepolypeptide, such as a receptor (e.g., an integrin, cytokine receptor,chemokine receptor or hormone receptor). In particular embodiments, thecytokine may be IL-12 or IL-15.

Yet a further aspect is a method for providing controlled expression ofa first transgene comprising providing a transgenic mammalian cellcomprising a nucleic acid comprising the first transgene operably linkedto a nucleotide sequence encoding TYMS^(SS), said cell furthercomprising a nucleotide sequence encoding DHFR^(FS). In someembodiments, the first transgene and nucleotide sequence encodingTYMS^(SS), upon expression, are encoded on the same mRNA. In furtherembodiments, the sequence encoding the first transgene and thenucleotide sequence encoding TYMS^(SS) are separated by an IRES or aribosomal slip sequence. In certain embodiments, the first transgene ofinterest is a growth factor, is a growth factor, a CAR construct, a TCR,a hormone (e.g., glucagon), a cytokine, a chemokine, a suicide gene, atranscription factor (e.g., FoxP3) or a cell surface polypeptide, suchas a receptor (e.g., an integrin, cytokine receptor, chemokine receptoror hormone receptor). In particular embodiments, the cytokine may beIL-12 or IL-15.

In further embodiments, the nucleotide sequence encoding DHFR^(FS) isoperably linked to a second transgene. In some embodiments, the secondtransgene and the nucleotide sequence encoding DHFR^(FS), uponexpression, are encoded on the same mRNA. In other embodiments, thesequence encoding the second transgene of interest and nucleotidesequence encoding DHFR^(FS) are separated by an IRES or a ribosomal slipsequence. In certain embodiments, the second transgene is a suicidegene. In specific embodiments, the suicide gene is an inducible suicidegene. In particular embodiments, the suicide gene is an inducibleCaspase 9. In some embodiments, the mammalian cell is a T-cell.

In another aspect is provided a recombinant nucleic acid moleculeencoding TYMS^(SS) and a first transgene coding sequence. In someembodiments, the sequence encoding TYMS^(SS) and/or the sequenceencoding the transgene of interest is codon optimized. In certainembodiments, recombinant nucleic acid is a DNA or a RNA (e.g., a mRNA).In some embodiments, the sequence encoding the transgene of interest andnucleotide sequence encoding TYMS^(SS) are separated by an IRES or aribosomal slip sequence. In some embodiments, the transgene of interestis a growth factor, is a growth factor, a CAR construct, a TCR, ahormone (e.g., glucagon), a cytokine, a chemokine, a suicide gene, atranscription factor (e.g., FoxP3) or a cell surface polypeptide, suchas a receptor (e.g., an integrin, cytokine receptor, chemokine receptoror hormone receptor). In particular embodiments, the cytokine may beIL-12 or IL-15.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are exemplary only, and should not be construed as limitingthe invention.

FIG. 1A depicts a pathway showing the role of synthesis of thymidine inDNA replication and cell survival.

FIG. 1B depicts the design of putative AThyR transgenes resistant toAThy toxicity in order to confer resistance to T cells that might beused in a combination therapeutic with AThy chemotherapy. AThyRs wereco-expressed with a fluorescent protein to indicate that surviving cellscontained the transgene. These transgene utilized the Sleeping Beautytransposon/transposase system to induce stable transgene expression inJurkat. Human muteins DHFR^(FS)-resistant to MTX (left), human muteinTYMS^(SS)—resistant to 5-FU (center), and the gold-standard Neomycinresistance gene (NeoR) drug resistance gene—resistance to G418 (right)were used in this study. Codon optimized (CoOp) versions of DHFR^(FS) &TYMS^(SS) replaced native codon DHFR^(FS) & TYMS^(SS) to test whetherknown post-transcriptional regulatory mechanisms were affecting AThyRselection or survival.

FIG. 1C depicts three different panels showing the percentage of eGFP+viable Jurkat T cells following treatment with MTX (left panel), 5-FU(center panel) and G418 (right panel) at varying concentrations. Theleft panel relates to DHFR^(FS)-2A-GFP (DG), CoOp DG, and no DNA, thatwere electroporated into Jurkat and subjected to MTX after 2 days. Thecenter panel relates to TYMS^(SS)-2A-GFP (TSG), CoOp TSG, and No DNAelectroporated Jurkat that were treated on day 2 with 5-FU. The rightpanel relates to NeoR-GFP and No DNA electroporated Jurkat that weretreated on day 2 with G418. For each experiment in C the percentage ofeGFP⁺ viable Jurkat is given after testing on day 8-10 after theaddition of drug.

FIG. 1D depicts the effect of MTX and Pemetrexid on the survival ofcells that expressed native DHFR and TYMS (“No DNA”) or expressed. DGand TYMS^(SS)-2A-RFP (TSR) were co-electroporated into Jurkat todetermine whether combination DHFR^(FS) & TYMS^(SS) confer enhancedsurvival to MTX (left) or Pemetrexid (right).

FIG. 1E depicts that following 2 weeks of selection in 1 μM MTX,[DHFR^(FS) & TYMS^(SS)]⁺ Jurkat displayed a uniform and repeatablepattern of correlated expression. Shown here, four separate [DHFR^(FS) &TYMS^(SS)]⁺ Jurkat experiments are overlaid in different shades.Experiments were independently repeated at least twice with 4-6replicates. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.;Dihydrofolate (DHF); DHF reductase (DHFR); deoxyuridine monophosphate(dUMP); deoxythymidine monophosphate (dTMP); 5,10-methylenetetrahydrofolate (5,10 CH₂THF); nicotinamide adeninedinucleotide phosphate (NADP).

FIG. 2A-I depicts experiments relating to viability of Jurkat cellsgiven for DHFR^(FS) (left), TYMS^(SS) (right), and NeoR (center).

FIG. 2A-II depicts experiments relating to alternations of meanfluorescent intensity (MFI) of eGFP given for DHFR^(FS) (left),TYMS^(SS) (right), and NeoR (center).

FIG. 2B depicts a determination whether enhanced survival occurs whenRaltitrexed and DHFR^(FS) & TYMS^(SS) were co-electroporated into Jurkattreated with Ral.

FIG. 2C depicts the correlation of expression of DHFR^(FS) and TYMS^(SS)plasmids that were independently expressed. Observations suggested thatcells expressing DHFR^(FS) & TYMS^(SS) as independent plasmids havecorrelated expression of each plasmid. This could have implications inthe co-regulation of DHFR^(FS) with TYMS^(SS). Hence, the MFI of eGFPand RFP were correlated for treatments with multiple concentrations ofMTX, Pem, and Ral. The linear regression data is included in the FIG.Each experiment was independently repeated at least twice with 4-6replicates. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. There wasobserved improved expression over mock electroporated Jurkat, and a weaksurvival improvement in 5 μM 5-FU. Without wishing to be bound bytheory, the lack of significantly enhanced survival is likely due to analternative mechanism of 5-FU contributing to toxicity, which is likelythe known inhibition of mRNA and rRNA synthesis by 5-FU. See Longley DB, et al., The Journal of biological chemistry 2010,285(16):12416-12425.

FIG. 3A depicts a propagation schematic showing initial AaPCstimulation. Two days after AaPC stimulation, the co-cultures received0.1 μM MTX, 5 μM 5-FU, or 1.4 mM G418 until day 14. The co-cultures werere-stimulated with AaPC at a 1:1 ratio and given 50 IU/mL IL-2 every 7days from day 1 to 35. Phenotypic changes in transgene expression weretracked during drug administration for the first 14 days and for the 21days after drug administration had ended

FIG. 3B-i shows the tracking of T cells for expression of AThyRsDHFR^(FS)-DG, TYMS^(SS)-TG, both [DG & TSR], and NeoR-NRG in thepresence (day 2-14) then absence (day 14-35) of appropriate selectiondrug. All experiments contain 5-6 biological replicates with eachexperiment independently repeated two times. *=p<0.05; **=p<0.01;***=p<0.001; ****=p<0.0001.

FIG. 3B-ii shows the percentage of T cells shown in FIG. 4B-I thatexpress co-receptor CD4.

FIG. 3C-i shows the tracking of T cells for expression ofMyc-ffLuc-2A-NeoR (NRF) combined with each AThyR transgene [DG & NRF],[TSG & NRF], and [DG & TSR & NRF] in order to improve selection forAThyRs selected by 5-FU. Selection occurred under the same condition asFIG. 4B-I, with the exception that 100 IU IL-2/mL was added to promoteoutgrowth of cells treated with G418. All experiments contain 5-6biological replicates with each experiment independently repeated twotimes. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

FIG. 3C-ii shows the percentage of T cells shown in FIG. 4C-I thatexpress co-receptor CD4.

FIG. 3D-i shows that to elucidate the influence of 5-FU and TYMS^(SS) onthe selection of DHFR^(FS), RFP or TYMS^(SS)-RFP (TSR) that wereco-electroporated into T cells with DHFR^(FS). All experiments contain5-6 biological replicates with each experiment independently repeatedtwo times. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

FIG. 3D-ii shows the percentage of T cells shown in FIG. 4D-I thatexpress co-receptor CD4.

FIGS. 4A-4C show the propagation characteristics of AThyR+ T cells inthe presence or absence of MTX, 5-FU, and/or G418. FIG. 4A shows AThyRand NeoR electroporated primary T cells were compared on Day 21 tomock-electroporated T cells treated with the same conditions. Eachexperiment was independently repeated at least twice with 5-6replicates. *=p<0.05, **=p<0.01. FIG. 4B, panel I depicts the continuedpropagation of the experiment of FIG. 5A on day 35. Each experiment wasindependently repeated at least twice with 5-6 replicates. *=p<0.05,**=p<0.01. FIG. 4B, panel II depicts the day 35 changes in outgrowthpotential for primary T cells when NeoR is combined with DHFR^(FS)and/or TYMS^(SS). Each experiment was independently repeated at leasttwice with 5-6 replicates. *=p<0.05, **=p<0.01. FIG. 4C shows theinfluence of 5-FU on preserving outgrowth potential for primary T cellson day 35. Each experiment was independently repeated at least twicewith 5-6 replicates. *=p<0.05, **=p<0.01.

FIGS. 5A-5H: Cis-transgenes downstream of DHFR^(FS) increase in thepresence of MTX independent of mRNA sequence and the increase issuppressed by restoration of thymidine synthesis. FIG. 5A Jurkat cellswere genetically-modified to express FLAG-DHFR^(FS)-2A-eGFP pSBSO(D^(FS)G) with resistance to MTX (n=4), codon optimized (CoOp)D^(FS)G—with known mRNA binding elements D^(FS)G removed (n=5), and[D^(FS)G & FLAG-TYMS^(SS)-2A-RFP pSBSO (TS^(SS)R)]— with enhancedresistance to MTX beyond D^(FS)G alone through the addition of MTXresistant TYMS^(SS) (n=7). Genetically-modified Jurkat cells wereselected for 2 weeks in 1 μM MTX before culturing without MTX for 3-5weeks. The stable fluorescent protein expression, in the absence of MTX,is depicted by mean fluorescence intensity (MFI). FIG. 5B-I Jurkat cellswere treated for 72 hours with 0.5 μM MTX or no treatment. The MFIdifference (Δ=eGFP MFI MTX treated—eGFP MFI untreated) is depicted. FIG.5B-II a representative histogram demonstrates the MTX induced change ineGFP MFI for DHFR^(FS) (left peak) and CoOp DHFR^(FS) (right peak) inJurkat. FIGS. 5C-D show in primary T cells, transgenes DHFR^(FS),TYMS^(SS), or the combination were selected for 2 weeks in the presenceof cytotoxic drug and then propagated without selection for 3 weeks (seeexamples). On day 35, T cells were stimulated with anti-CD3, anti-CD28antibodies, and 50 IU/mL IL-2 in the presence or absence of MTX. Thefluorescent protein MFI of untreated cells is shown in FIG. 5C, and FIG.5D-I depicts the A MFI after 72 hours of treatment with 0.5 μM MTX incomparison to no treatment. FIG. 5D-II, shows a representativehistogram, which demonstrates the observed shift in eGFP fluorescencefor DHFR^(FS)+ T cells in the presence or absence of MTX (n=5). (NoDNA=far left peak; D^(FS)G & NRF, No Trx=upper center peak; D^(FS)G &NRF, MTX=upper right peak; D^(FS)G & TS^(SS)R, No Trx=lower center andlower right peak; D^(FS)G & TS^(SS)R, MTX=lowest peak). FIG. 5E, a transregulatory pattern of DHFR and TYMS linked fluorescent proteins wasobserved. A representative flow plot from the 1 μM MTX selected Jurkatleft untreated in (5A) demonstrates that unselected mock-electroporated(No DNA—lower left cluster) Jurkat and D^(FS)G+ Jurkat (lower rightcluster) have a globular appearance in the RFP channel, whileco-expression of DHFR^(FS) with TYMS^(SS) in [D^(FS)G & TS^(SS)R]+Jurkat leads to a linear clustering (upper right cluster). FIG. 5F Tcells were electroporated with DHFR^(FS) and co-transformed with eitherRFP control or FLAG-TYMS^(SS)-2A-RFP pSBSO (TS^(SS)R) before propagationas before (in 5C) with selection in 0.1 μM MTX from days 2-14 beforecontinued propagation in the absence of MTX. A representative flow plotof primary human T cells from the same donor where [D^(FS)G & RFP(cluster on the far right)], [D^(FS)G & TS^(SS)R (upper right cluster)],and untransformed T cells (lower left quadrant) are shown on day 21. Alinear clustering of DHFR^(FS) is again noted when co-expressed withTYMS^(SS) that is not noted with RFP alone. FIG. 5G further studies toidentify a trans pattern of linked expression between DHFR^(FS) andTYMS^(SS) were identified in the selection of [D^(FS)G & TS^(SS)R]electroporated Jurkat in anti-folates MTX [0, 0.01, 0.1, 0.5, 1, 5 μM],pemetrexed [0, 10, 50, 100 μM], and raltitrexed [0, 1, 5, 10 μM]. TheMFI of D^(FS)G and TS^(SS)R for each expression pattern was plottedafter day 2-14 in selection. The values are plotted and a linear fittingwas performed with the R² from the Pearson's correlation and the slopeof the linear regression provided on the graph. This data is assembledfrom 4 technical replicates. FIG. 5H depicts a model ofpost-transcriptional regulation of DHFR and TYMS. All experiments otherthan that depicted in FIG. 5G were independently repeated twice.Kruskall-Wallis test was used to determine significant differences withmultivariate analyses; *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.TMP—thymidine monophosphate; UMP—uridine monophosphate;DHF—dihydrofolate; THF—tetrahydrofolate; 5, 10-methylenetetrahydrofolate(5, 10 CH2THF).

FIGS. 5I-5L, shows co-expression of DHFR^(FS) with TYMS^(SS) leads tocontrolled expression of TYMS^(SS) and cis transgenes in the presence ofMTX. FIGS. 5I-5J, T cells from the experiment described in FIG. 5F werepropagated to day 35. T cells were stimulated for 72 hours withanti-CD3, anti-CD28 antibodies, 50 IU/mL IL-2, and varyingconcentrations of MTX. The MTX induced change in eGFP MFI for DHFR^(FS)is shown in (5I), while the influence of MTX on RFP and RFP co-expressedwith TYMS^(SS) (TS^(SS)R) is shown in (5J) (n=6, repeated independentlytwice, analyzed by Two-Way ANOVA with Sidak's multiple comparison test).FIG. 5K, this regulatory pattern was applied to a clinically relevantproblem: The cytokine interleukin-12 (IL-12) is a strong promoter ofanti-tumor activity in T cells, but is highly toxic. A constructexpressing IL-12 following TYMS^(SS), called TS^(SS)IL-12, was used tomodulate IL-12 expression in conjunction with the construct D^(FS)iC9.D^(FS)iC9 is capable of selecting T cells with DHFR^(FS) or depleting Tcells with inducible caspase 9 (iC9). A representative flow diagram ofthe same donor depicts intracellular expression of IL-12 and c-Myc-iC9in [DFSiC9 & TS^(SS)IL-12]-expressing T cells. These cells are shown onday 21 after selection from day 2-14 in 0.1 μM MTX and subsequenttreatment with 0.5 μM MTX (right cluster) or no treatment (left cluster)from days 14-21. Cellular excretion of IL-12 was blocked for 6 hoursbefore intracellular staining. Gating is based on staining ofuntransformed, unselected T cells stained in the same way. FIG. 5L,three donors were treated as in (K) and the change in transgeneexpression noted after 7 days of treatment with 0.5 μM MTX is shown.Each measure was analyzed by t-tests. ns=not significant; *=p<0.05,**=p<0.01, ***=p<0.001, ****=p<0.0001.

FIGS. 6A-6C depict flow plots of transgene expression for AThyRexperiments on day 35. Flow plots of CD4 and GFP expression depict day35 of a series of experiments designed to characterize the selection andmaintenance of transgene expression in donor T cells. T cells grown for35 days with days 2-14 in the presence of cytotoxic drugs MTX, 5-FU,G418, or a combination, as noted above the flow plot. FIG. 6A depictsexperimental conditions that corresponds to the experiment described forFIG. 3B. FIG. 6B depicts experimental conditions that corresponds to theexperiment described for FIG. 3C. FIG. 6C depicts experimentalconditions that corresponds to the experiment described for FIG. 3D.

FIG. 6D shows that the presence of ffLuc-2A-NeoR—NRF—on day 35 forexperiment noted in FIG. 6B is demonstrated using D-luciferin to induceT cell chemiluminescence. Each experiment was independently repeated atleast twice with 6 replicates. Representative flow plots are depicted.*=p<0.05, **=p<0.01, ***=p<0.001.

FIGS. 7A-7C depict AThyR rescue of AThyR⁺ and AThyR^(neg) T cellsfollowing 72 hours treatment in MTX. T cells from the experimentdescribed for FIG. 3D were stimulated on day 35 with anti-CD3,anti-CD28, and IL-2 along with varying doses of MTX [0, 0.1, 0.5, 1 μM]for 72 hours. FIG. 7A shows the gating strategy and representative flowplots. FIG. 7B shows enhanced viability of AThyR+ T cell cultures. FIG.7C shows assessment of Viable, CD3⁺, GFPneg, RFP^(neg) T cells(AThyR^(neg)) for survival. Each experiment was independently repeatedat least twice with 6 biologic replicates total. Representative flowplots from one are depicted; ns=no significance; *=p<0.05, **=p<0.01,***=p<0.001; ****=p<0.0001.

FIGS. 8A-8E depict an example that AThyRs select for transgenes ofinterest. Increased selection of DHFR^(FS) is desirable for difficult toisolate genes of interest, such as suicide genes. FIG. 8A shows aconstruct in which the suicide gene inducible caspase 9 (iC9) wasdesigned to express with DHFR^(FS) in the plasmid DFSiC9 shown in (A).FIG. 8B shows the testing of the construct depicted in FIG. 8A in PBMCof 3 healthy donors stimulated with a 1:1 ratio of OKT3-loaded AaPC andtreated with MTX from day 2 until day 7 when survival is shown. FIG. 8Cshows T cells were electroporated with CD19-specific chimeric antigenreceptor (CAR), D^(FS)iC9, and SB transposase and expanded on CARL⁺ K562in the presence of MTX for 21 days to select for each transgene, withCARL an acryonym for ligand for CAR. The expression of costimulatory Tcell receptors CD4, CD8, and transgenes CAR and DHFR^(FS) are shown in21 day CARL expanded transgenic T cells in comparison to mockelectroporated T cells expanded on OKT3-loaded AaPC clone.4. Experimentswere performed with 4 normal donors and repeated twice. Significance foreach comparison was initially determined by Two-Way ANOVA followed bySidak's post-hoc analysis; *=p<0.05, **=p<0.01, ***=p<0.001,****=p<0.0001. FIG. 8D shows the effect of MTX on cytotoxicity inDHFR^(FS+) CAR⁺ T cells was tested by stimulating CAR⁺ T cells in thepresence or absence of MTX for 7 days after stimulation on day 14.Cytotoxicity was assessed by chromium release assay (CRA) on Day 21using CD19 positive or CD19 negative murine lymphoma EL-4 cells. T cellswere co-incubated with EL-4 at a 1 target:5 effector ratio. Experimentswere performed with 4 normal donors and repeated twice. Significance foreach comparison was initially determined by Two-Way ANOVA followed bySidak's post-hoc analysis; *=p<0.05, **=p<0.01, ***=p<0.001,****=p<0.0001. FIG. 8E shows the assessment of the functionality of iC9on day 21 by resting T cells for 48 hours in 10 nM AP20187. T cells hadpreviously been stimulated for 7 days in the presence or absence of MTX.Comparison of surviving CAR⁺ T cells is made to matched, un-treatedcells. Experiments were performed with 4 normal donors and repeatedtwice. Significance for each comparison was initially determined byTwo-Way ANOVA followed by Sidak's post-hoc analysis; *=p<0.05,**=p<0.01, ***=p<0.001, ****=p<0.0001. Co-expressing DHFR^(FS) with iC9rather than CAR added the potential to ablate T cells through theaddition of iC9 chemical inducer of dimerization AP20187. The additionof AP20187 significantly depleted resting CAR⁺ T cells independent ofMTX. This demonstrates that D^(FS)iC9 can select for iC9 expression anddeplete genetically-modified T cells as necessary. The use of DHFR^(FS)has the advantage of selecting transgene expression in T cellsindependent of antigen-specificity and antigen expression, makingDHFR^(FS) a more portable tool for use in a variety of T cell studies.

FIG. 9 depicts that post-transcriptional regulation of thymidinesynthesis locks expression of DHFR to TYMS. MTX-induced increases inDHFR expression were inhibited by restoration of thymidine synthesis(TMP—thymidine monophosphate from UMP—uridine monophosphate). Likewise,MTX-induced decreases in TYMS expression were restored to normal levelsby the restoration of DHFR activity reducing DHF—dihydrofolate toTHF—tetrahydrofolate.

FIGS. 10A-10D show that the drug selection of T_(CD4, FoxP3) by MTXoccurs in part through toxicity. The known selection of T_(CD4, FoxP3)by MTX was analyzed by targeting enzymes that contribute to the actionof MTX. As T_(CD4, FoxP3) are a rare component of PBMC, drug basedinhibition was originally sought to analyze the phenomenon. Multipledrugs with actions similar to MTX were used to assay for the selectionof T_(CD4, FoxP3). In this case, γ-irradiation, G418, and cisplatin(CDDP) were used for controls as none of those treatments act on theknown enzymatic targets of MTX. FIG. 10A shows the association of eachdrug to the enzyme targets of MTX. FIG. 10B, panel I shows PBMCstimulated with anti-CD3/CD28 and soluble human IL-2 were given lethaldoses of each treatment and assayed after 7 days for viability. FIG.10B, panel II shows that these treatments resulted in variable selectionfor T_(CD4, FoxP3) on day 7. The inability of folate analogs targetingDHFR, TYMS, or GARFT to significantly select for T_(CD4, FoxP3)suggested that inhibition of AICARtf/inosine monophosphate (IMP)cyclohydrolase (ATIC) contributes to this selection. A dose dependencestudy followed analyzing the contribution of ATIC inhibitor in theselection of T_(CD4, FoxP3). The study in B-II noted that G418 depletedT_(CD4, FoxP3), thus, this was used as a negative control while theknown selection of T_(CD4, FoxP3) by rapamycin (Rapa) was a positivecontrol. A non-folate analog known to inhibit ATIC (iATIC) was used as aspecific inhibitor of ATIC. FIGS. 10C and 10D show the cytotoxicity ofG418 and MTX.

FIGS. 10E and 10F-i show the cytotoxicity of iATIC and Rapa.

FIG. 10F-ii (four panels) shows the selection for TCD4, FoxP3 for G418,MTX, iATIC, and Rapa.

FIG. 10G depicts flow plots for CD4 and FoxP3 expression. FoxP3expression was enhanced by iATIC similar to the action of Rapa,suggesting that MTX selection relies in part on cytotoxicity and in partby inhibition of ATIC to enhance selection of T_(CD4, FoxP3). All assaysused 4-7 donors independently repeated 2-3 times. Statisticalsignificance was assessed using One-Way ANOVA for viability andKruskall-Wallis test for percentage of T_(CD4, FoxP3); *=p<0.05,**=p<0.01, ***=p<0.001, ****=p<0.0001.

FIGS. 11A-11B show correlative findings in the selection of Tregs fromprimary T cells through resistance to the anti-DHFR and anti-TYMSactions of MTX. FIG. 11A shows the selection of TCD4, FoxP3 was assessedat day 21 in each experiment. Selection of TCD4, FoxP3 was assessed atday 21 in each experiment. The selection of T_(CD4, FoxP3) in theexperiment corresponding to column I of FIG. 2 is shown in A. It isnotable for the rescue of T_(CD4, FoxP3) with NeoR and early selectionof T_(CD4, FoxP3) with MTX selection of DHFR^(FS). FIG. 11B shows flowplots in which FoxP3 is co-expressed with IL-2 in the top row, LAP inthe middle row or CTLA-4 in the bottom row for the same experiment afterstimulation on Day 35. This experiment utilized 5 donors and wasindependently repeated twice. Significance was assessed by Two-Way ANOVAand Sidak's post-hoc; *=p<0.05, **=p<0.01.

FIGS. 12A-12D show that primary T cells resistant to the anti-DHFR andanti-TYMS actions of MTX preferentially expand Tregs. Primary T cellswere electroporated with DHFR^(FS) and TYMS^(SS) transgenes resistant tothe anti-DHFR and anti-TYMS actions of MTX, respectively, in order toassess the contribution of each pathway to the selection ofT_(CD4, FoxP3). T cells were electroporated with plasmids expressingdrug resistant transgenes and stimulated with artificial antigenpresenting cells (AaPCs) weekly at a 1:1 ratio. T cells were selectedfor 2 weeks in the combined with TYMSSS-2A-RFP (TSR) and selected usingboth MTX and SFU, or control selection vector NeoR-2A-GFP (NRG) selectedwith G418. Selection of TYMS_(SS) by 5-FU was incomplete. Thus,ffLuc-2A-NeoR (NRF) vector was included with the MTX resistanttransgenes DG, TSG, or [DG & TSR] to remove untransformed T cells in theexperiments shown in column II. Equivalent selection for each transgeneshowed that MTX enhanced selected for T_(reg) in the presence of MTXresistant DHFR. It was still uncertain whether the enzymatic activity ofTYMS or 5-FU played a part in the selection of T_(reg). Therefore, theexperiment shown in column III was performed to test the influence ofTYMS inhibition in the selection of T_(reg). Selection of T_(reg)phenotype was found to be associated with 5-FU, but independent of TYMSactivity. The Kruskall-Wallis test was used to assess differencesbetween groups for 5-6 biologic replicates and tests were independentlyrepeated twice; *=p<0.05, **=p<0.01.

FIG. 13 is a diagrammatic representation of biochemical and proteininteractions thought to influence selection of T_(reg).

FIGS. 14A-14E show that ribosomal Inhibition by aminoglycoside G418selectively depletes replicating T_(CD4, FoxP3). FIG. 14A shows thatthawed PBMC were stimulated with anti-CD3/CD28 and IL-2 in the presenceof increasing concentrations of G418, hygromycin, zeocin, or rapamycinfor 7 days and the selection for T_(CD4, FoxP3). FIG. 14B shows flowplots of FoxP3 and CD4 expression, which in turn show the representativetrends for one donor following the use of each drug. FIG. 14C, the toppanel shows the loss of T_(CD4, FoxP3) was tested in un-stimulated,thawed PBMC over the course of 9 days with or without G418 while thebottom panel shows the effects of G418 on proliferating andnon-proliferating T_(CD4, FoxP3) as indicated by Ki-67. In FIG. 14D,representative flow plots for one donor demonstrate the effect of G418on CD4 and FoxP3 expression in the top panel while FoxP3 and Ki-67expression are shown in the bottom panel. Gentamicin is an FDA approvedaminoglycoside antibiotic and was subsequently tested in comparison toG418 for depletion of T_(CD4, FoxP3) over a 7 day period. Allexperiments were performed with 6 normal donors and repeatedindependently twice. FIG. 14E depicts the depletion of T_(CD4, FoxP3) inresting PBMC after 7 days from gentamicin, an aminoglycosin, anddemonstrates the action of aminoglycosides in depleting T_(CD4, FoxP3).It was next tested whether depletion of T_(CD4, FoxP3) corresponded witha loss of T_(reg) marker expression or selective T_(reg) toxicity.

FIGS. 15A-15D show the effects of MTX, 5-FU, and G418 in sorted T_(reg).FIG. 15A diagrammatically shows the T_(reg) and T_(eff) were treatedwith MTX, 5-FU, or G418 as before for 7 days before stimulating withoutdrug for the remaining 2 weeks of the experiment. FIG. 15B shows anassessment of markers and activity of T_(reg) on Day 21 to determine thecontribution of each drug to selection or depletion of T_(reg), and thelive T_(CD4, FoxP3) on Day 21 are shown in B. FIG. 15C shows that afterstimulating with soluble anti-CD3/CD28 and IL-2 for 48 hours T cellswere assessed for co-expression of FoxP3 with CD25 in C-I, FoxP3 withCTLA-4 in C-II, and FoxP3 with LAP in C-IV. Six hours of stimulationwith PMA/ionomycin was used to assess loss of IL-2 secretion in FoxP3expressing T cells, C-III. A 72 hour suppression assay was performed bymixing treated T_(reg) with untreated T_(eff) and looking at uptake of[³H] Thymidine at two separate concentrations, shown in D. Thisexperiment was performed with 5 normal donors and repeated twice. Allexperiments were assessed with Two-Way ANOVA and significance wasdetermined by Sidak's post-hoc analysis; *=p<0.05, **=p<0.01,***=p<0.001, ****=p<0.0001.

FIGS. 16A-E show that stimulation of T_(CD4, FoxP3) enhances adenosinemonophosphate (AMP) Kinase (AMPK) activation and leads to inhibition oftranslational elongation factor eEF2. Differentiation of T_(CD4, FoxP3)from CD4+CD25_(neg) T cells was accomplished by gating in the stimulatedand unstimulated experiments. FIG. 16A depicts the mean fluorescenceintensity (MFI) of AMPK activated by phosphorylation at T172 afterstimulation in the top panel while the lower panel of FIG. 16A depictsthe MFI of activated S6 by phosphorylation at sites S235/S236. FIG. 16Bdepicts a flow plot depicting the changes in phosphorylation forT_(CD4, FoxP3) and CD4⁺ CD25_(neg) T cells in the upper panel for AMPKand in the lower panel for S6 with respect to FoxP3 expression in gatedCD4⁺ cells. FIG. 16C is an image cytometry gallery depicting fluorescentand morphologic changes in T_(CD4, FoxP3) following stimulation. FIG.16D shows an image cytometer was used to analyze p-eEF2 T56 MFI anddepicts an increase in activation of T_(CD4, FoxP3). FIG. 16E shows thedifference from CD4⁺ FoxP3^(neg) T cells in image cytometry gallery.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art. The following terms are provided below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. Thus, recitation of “a cell”, for example, includes aplurality of the cells of the same type.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of +/−20% or +/−10%, more preferably +/−5%, even morepreferably +/−1%, and still more preferably +/−0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

By “animal” is meant any member of the animal kingdom includingvertebrates (e.g., frogs, salamanders, chickens, or horses) andinvertebrates (e.g., worms, etc.). “Animal” is also meant to include“mammals.” Preferred mammals include livestock animals (e.g., ungulates,such as cattle, buffalo, horses, sheep, pigs and goats), as well asrodents (e.g., mice, hamsters, rats and guinea pigs), canines, felines,primates, lupine, camelid, cervidae, rodent, avian and ichthyes.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab fragments and F(ab)₂ fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies (mAb), chimeric antibodies and humanizedantibodies. The production of antibodies and the protein structures ofcomplete, intact antibodies, Fab fragments and F(ab)₂ fragments and theorganization of the genetic sequences that encode such molecules arewell known and are described, for example, in Harlow et al., ANTIBODIES:A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1988) which is incorporated herein by reference.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

By “epitope” is meant a region on an antigen molecule to which anantibody or an immunogenic fragment thereof binds specifically. Theepitope can be a three dimensional epitope formed from residues ondifferent regions of a protein antigen molecule, which, in a nativestate, are closely apposed due to protein folding. “Epitope” as usedherein can also mean an epitope created by a peptide or hapten portionof matriptase and not a three dimensional epitope.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

As used herein, the term “fusion protein” or “fusion polypeptide” is apolypeptide comprised of at least two polypeptides and optionally alinking sequence, and that are operatively linked into one continuousprotein. The two polypeptides linked in a fusion protein are typicallyderived from two independent sources (i.e., not from the same parentalpolypeptide), and therefore a fusion protein comprises two linkedpolypeptides not normally found linked in nature. Typically, the twopolypeptides can be operably attached directly by a peptide bond, or maybe connected by a linking group, such as a spacer domain. An example ofa fusion polypeptide is a polypeptide that functions as a receptor foran antigen, wherein an antigen binding polypeptide forming anextracellular domain is fused to a different polypeptide, forming a“chimeric antigen receptor”.

By “knock-in” of a target gene means an alteration in a host cell genomethat results in altered expression (e.g., increased, including ectopic)of the target gene, e.g., by introduction of an additional copy of thetarget gene or by operatively inserting a regulatory sequence thatprovides for enhanced expression of an endogenous copy of the targetgene. See U.S. Pat. No. 6,175,057.

By “knock-out” of a gene means an alteration in the sequence of the genethat results in a decrease of function of the target gene, preferablysuch that target gene expression is undetectable or insignificant. SeeU.S. Pat. No. 6,175,057.

By “modulating” or “regulating” is meant the ability of an agent toalter from the wild type level observed in the individual organism theactivity of a particular gene, protein, factor, or other molecule.

By “mutant” with respect to a polypeptide or portion thereof (such as afunctional domain of a polypeptide) is meant a polypeptide that differsin amino acid sequence from the corresponding wild type polypeptideamino acid sequence by deletion, substitution or insertion of at leastone amino acid. A “deletion” in an amino acid sequence or polypeptide isdefined as a change in amino acid sequence in which one or more aminoacid residues are absent as compared to the wild-type protein. As usedherein an “insertion” or “addition” in an amino acid sequence orpolypeptide is a change in an amino acid sequence that has resulted inthe addition of one or more amino acid residues as compared to thewild-type protein.

As used herein “substitution” in an amino acid sequence or polypeptideresults from the replacement of one or more amino acids by differentamino acids, respectively, as compared to the wild-type protein.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used, “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The term “linker”, also referred to as a “spacer” or “spacer domain” asused herein, refers to a an amino acid or sequence of amino acids thatthat is optionally located between two amino acid sequences in a fusionprotein.

The term “operably linked” (and also the term “under transcriptionalcontrol”) refers to functional linkage between a regulatory sequence anda heterologous nucleic acid sequence resulting in expression of thelatter. For example, a first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to a human being.

The term “polynucleotide” is a chain of nucleotides, also known as a“nucleic acid”. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, and include both naturally occurring and syntheticnucleic acids.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” means a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

By “somatic cell” is meant any cell of a multicellular organism,preferably an animal, that does not become a gamete.

The term “therapeutically effective amount” shall mean that amount ofdrug or pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system or animal that is being sought by aresearcher or clinician.

The term “transfected” or “transformed” or “transduced means to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The transduced cell includes the primary subjectcell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Examples of vectors include but are notlimited to, linear polynucleotides, polynucleotides associated withionic or amphiphilic compounds, plasmids, and viruses. Thus, the term“vector” includes an autonomously replicating plasmid or a virus. Theterm is also construed to include non-plasmid and non-viral compoundswhich facilitate transfer of nucleic acid into cells, such as, forexample, polylysine compounds, liposomes, and the like. Examples ofviral vectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Where any amino acid sequence is specifically referred to by a SwissProt. or GENBANK Accession number, the sequence is incorporated hereinby reference. Information associated with the accession number, such asidentification of signal peptide, extracellular domain, transmembranedomain, promoter sequence and translation start, is also incorporatedherein in its entirety by reference.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one aspect, an isolated transgenic mammalian T cell comprising orexpressing a transgene and one or more of DHFR^(FS) and TYMS^(SS) isprovided. In some embodiments, the isolated transgenic mammalian T cellcomprises or expresses a transgene, DHFR^(FS) and TYMS^(SS). Briefly, Tcells can be obtained from peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Tcell lines available in the art may be used. Preferably, T cells areobtained from a unit of blood collected from a subject using any numberof techniques known to those skilled in the art. Isolation of T cellsmay proceed according to procedures known in the art, as described inUS2013/0287748 A1. The harvested T cells are then expanded using methodswell-known in the art, such as described in US2013/0287748 A1.

According to one embodiment, T-cells are harvested and processed forlentiviral transduction as follows. Patient peripheral blood mononuclearcells are purified and washed in phosphate-buffered saline (PBS) with 1%human serum albumin. Lymphocytes are enriched using magnetic beaddepletion of monocytes, according to known methods. Lymphocytes arecultured according to Good Manufacturing Practice regulations aspreviously described by Levine et al., (1998), J Hematother 7:437-448.The cells are expanded ex vivo for 14 days in a serum-free hematopoieticcell medium, e.g., X-VIVO 15 of Lonza Group Ltd. (a chemically defined,serum-free hematopoietic cell medium) supplemented with 10% Normal HumanAntibody Serum, and then processed for reinfusion on day 14 ofculturing. The magnetic beads are removed using a magnetic cellseparation system. The cells are harvested, washed and resuspended in aPlasmalyte A containing 1% human serum albumin before being transducedwith lentiviral vectors.

As demonstrated herein, T cells are genetically modified to expressanti-thymidylate resistance (AThyR) transgenes, and other transgenes.AThyRs are shown to rescue T cells from anti-thymidylate (AThy) drugtoxicity, such as AThy toxicity mediated by 5-FU and anti-folatestargeting DHFR and TYMS. Also, as demonstrated herein DHFR muteins suchas DHFR^(FS) permits methotrexate (MTX)-inducible increase in transgeneexpression that is thymidine dependent, and TYMS muteins such asTYMS^(SS) permit MTX-inducible decrease in transgene expression that isdihydrofolate dependent. As further demonstrated herein, AThyRs can beused to positively select for transgenes of interest without the use ofimmunogenic genes or magnetic selection.

The use of AThyR transgenes DHFR^(FS) and TYMS^(SS) alone or incombination, engineered into T cells expressing a transgene of interest,provides a unique capacity to select for transgene expression within thebulk population, can modulate the expression of cis as well as transtransgenes of interest, and promote survival in toxic concentrations ofAThys. Thus, T cells expressing transgenes of interest, such as T cellsexpressing tumor-targeting chimeric antigen receptors (CARs), furtherengineered to express AThyRs such as DHFR^(FS) and/or TYMS^(SS), findutility in treating cancers such as lung, colon, breast, and pancreasthat are in need of new therapeutic options.

As demonstrated herein, combining AThyRs DHFR^(FS) and TYMS^(SS) in Tcells leads to significant survival advantages for such cells treatedwith toxic concentrations of AThys: MTX, Pem, or 5-FU. These AThy drugsare regularly used to treat lung and colon cancer among other commoncancers. The findings described herein indicate that AThyRs T cells cansurvive toxic AThy concentrations. Combining the immunomodulatoryeffects of chemotherapy like 5-FU with T cells resistant to thecytotoxic effects of 5-FU could substantially improve the anti-cancerresponse of the patient beyond that of either therapeutic used alone.

As described herein, for the purpose of selecting transgenes of interestfor T cell expression, AThyRs were compared to one of the earliest drugresistance transgenes—NeoR. As described herein, it was found thatDHFR^(FS) is superior to NeoR in promoting survival, selection, anddrug-dependent increases of expression of a representative transgene(eGFP). Notably, DHFR^(FS) and TYMS^(SS) have lower immunogenicity ashuman proteins, and MTX can be used both in vitro and in vivo¹ toimprove transgene selection, whereas G418 cannot. The findings describedherein demonstrate that DHFR^(FS) can select for cells expressingtransgenes such as the suicide gene iC9. Thus, DHFR^(FS) and [DHFR^(FS)& TYMS^(SS)] are attractive alternatives to alternative to magneticbeads for selecting T cells expressing one or more transgenes ofinterest. In fact, the potential to select for AThyR+ T cells in vivousing MTX indicates that transgene selection could be performed withinthe patient rather than ex vivo.

In another aspect is provided a method for inhibiting AThy toxicity in amammalian T cell comprising expressing an AThyR transgene in saidmammalian T cell. In some embodiments, the AThyR transgene is DHFR^(FS).In some embodiments, the AThyR transgene is TYMS^(SS).

In another aspect is provided a method for selecting a T cell expressinga transgene of interest. The method comprises applying a thymidinesynthesis inhibitor to a plurality of T cells that comprises a T cellexpressing the transgene of interest and DHFR^(FS) and selecting for oneor more T cells surviving after seven or more days of application of thethymidine synthesis inhibitor, wherein the one or more T cells expressesthe vector comprising the transgene of interest and DHFR^(FS). Thethymidine synthesis inhibitor may be selected from the group consistingof methotrexate (MTX), 5-FU, Raltitrexed and Pemetrexed.

In some embodiments, a DNA sequence, including DNA sequences from genesdescribed herein, is inserted into the vector. Vectors derived fromretroviruses are preferred, as they provide long-term gene transfersince and allow stable integration of a transgene and its propagation indaughter cells. Expression of nucleic acids encoding the AThyRsdescribed herein may be achieved using well-known molecular biologytechniques by operably linking a nucleic acid encoding the AThyRs to apromoter, and incorporating the construct into a suitable expressionvector. The vectors can be suitable for replication and integrationeukaryotes. Typical cloning vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence.

In some embodiments, one or more DNA constructs encode the transgene andone or more DNA constructs encoding one or more AThyRs, DHFR^(FS) andTYMS^(SS). In other embodiments, the transgene and the one or moreAThyRs, DHFR^(FS) and TYMS^(SS) are operably linked. A chimericconstruct encoding the various nucleotide sequences encoding one or moretransgenes and one or more AThyRs, DHFR^(FS) and TYMS^(SS) may beprepared by well-known molecular biology techniques, from naturallyderived or synthetically prepared nucleic acids encoding the components.The chimeric constructs may be prepared using natural sequences. Thenatural genes may be isolated and manipulated as appropriate so as toallow for the proper joining of the various domains. Thus one mayprepare the truncated portion of the sequence by employing polymerasechain reaction (PCR) using appropriate primers which result in deletionof the undesired portions of the gene. Alternatively, one may use primerrepair where the sequence of interest may be cloned in an appropriatehost. In either case, primers may be employed which result in terminiwhich allow for annealing of the sequences to result in the desired openreading frame encoding the CAR protein. Thus, the sequences may beselected to provide for restriction sites which are blunt-ended or havecomplementary overlaps. Preferably, the constructs are prepared byoverlapping PCR.

As demonstrated herein, anti-thymidylates or thymidine synthesisinhibitors, exemplified by MTX, can be used to regulate transgeneexpression either to higher or lower expression levels for a transgeneexpressed cis to DHFR^(FS) or TYMS^(SS). MTX-inducible positive ornegative modulation of cis-transgenes is believed clinically useful insituations where MTX is used to modulate the spatial and temporalexpression of dangerous but necessary transgenes in T cells, such astransgenes expressing certain chimeric antigen receptors (CAR) orcytokines. The correlated expression of DHFR^(FS) with trans expressedTYMS^(SS) is also useful in expressing proteins such as TCR α and β thatneed to be expressed at nearly equivalent amounts and where the use of2A mediated cleavage sites may adversely affect protein structure andfunction.

Yet another aspect is a method for selectively propagating peripheralblood mononuclear cells (PBMC) resistant to MTX and 5-FU. The methodcomprises transfecting peripheral PBMC with a vector comprising an AThyRgene, treating the transfected PBMC with a thymidine synthesis inhibitorand selecting for PBMC that express the AThyR gene. In some embodimentsof this aspect, the method further comprises propagating a T cellpopulation from the transfected PBMC. In some embodiments, the thymidinesynthesis inhibitor may be selected from the group consisting ofmethotrexate (MTX), 5-FU, Raltitrexed and Pemetrexed. In someembodiments, the thymidine synthesis inhibitor is MTX. In someembodiments, the AThyR gene is TYMS^(SS). In some embodiments, the AThyRgene is DHFR^(FS).

Another aspect is an isolated transgenic mammalian T cell comprising anucleic acid sequence comprising a transgene of interest and anucleotide sequence encoding DHFR^(FS) or TYMS^(SS). In someembodiments, the isolated transgenic mammalian T cell comprises anucleic acid comprising a transgene of interest and a nucleotidesequence encoding DHFR^(FS), wherein the transgene of interest and thenucleotide sequence encoding DHFR^(FS) are operably linked. In someembodiments, the isolated transgenic mammalian T cell comprises anucleic acid comprising a transgene of interest and a nucleotidesequence encoding TYMS^(SS), wherein the transgene of interest and thenucleotide sequence encoding TYMS^(SS) are operably linked.

In another aspect is provided an isolated transgenic mammalian T cellexpressing a transgene and DHFR^(FS), wherein the T cell comprises (1) apolynucleotide comprising sequence that encodes the transgene and (2) apolynucleotide comprising sequence that encodes the DHFR^(FS).

In certain aspects, a sequence encoding DHFR^(FS) encodes a polypeptideat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% to SEQ ID NO: 12. In some embodiments, a sequence encodingDHFR^(FS) encodes a polypeptide having no more than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 amino acid deletions, insertions or substitutions relative toSEQ ID NO: 12.

In another aspect is provided an isolated transgenic mammalian T cellexpressing a transgene and TYMS^(SS), wherein said T cell comprises (1)a polynucleotide comprising sequence that encodes the transgene and (2)a polynucleotide comprising sequence that encodes the TYMS^(SS).

In certain aspects, a sequence encoding TYMS^(SS) encodes a polypeptideat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% to SEQ ID NO: 11. In some embodiments, a sequence encodingTYMS^(SS) encodes a polypeptide having no more than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 amino acid deletions, insertions or substitutions relative toSEQ ID NO: 11.

In a further aspect, a composition is provided comprising a plurality ofhuman cells (e.g., T-cells), wherein the cells comprise a sequenceencoding TYMS^(SS) and a first transgene, said cells having been treatedwith MTX (e.g., in culture or in a living organism), thereby changingexpression of the transgene. In certain embodiments, the transgeneencodes a CAR, TCR, polypeptide hormone (e.g., an endocrinologicalhormone, such as glucagon), cytokine, a transcription factor orchemokine. In still further aspects, a transgene of the embodimentsencodes a cell surface polypeptide, such as an integrin, cytokinereceptor, chemokine receptor or a receptor of a hormone (e.g., aneurological or endocrine hormone).

In still a further aspect, a composition is provided comprising aplurality of human cells (e.g., T-cells), wherein the cells comprise asequence encoding DHFR^(SS) and a first transgene, said cells havingbeen treated with MTX (e.g., in culture or in a living organism),thereby changing expression of the transgene. In certain embodiments,the transgene encodes a CAR, TCR, polypeptide hormone (e.g., anendocrinological hormone, such as glucagon), cytokine, transcriptionfactor or chemokine. In still further aspects, a transgene of theembodiments encodes a cell surface polypeptide, such as an integrin,cytokine receptor, chemokine receptor or a receptor of a hormone (e.g.,a neurological or endocrine hormone).

In a further aspect, there is provided a composition comprising a firstplurality of T cells isolated from a mammal and treated with a thymidinesynthesis inhibitor, wherein the first plurality of T cells is enrichedfor regulatory T cells as compared to a second plurality of T cellsisolated from a mammal that is depleted by a thymidine synthesisinhibitor during stimulation with a(n) antibody(ies) compromising anysingular or combination use of anti-CD2, anti-CD3, anti-CD27, anti-CD28,anti-41BB, anti-OX40, phytohemagluttinin (PHA), ionomycin or peptidepulsed antigen presenting cells (whether synthetic or biologic and ofany cell origin whether human or otherwise if utilized to stimulate Tcells in such a way that the T cells begin to replicate).

In yet another aspect is provided a method of treating a patient with acancer comprising to administering to a patient a therapeuticallyeffective amount of a T cell of an isolated T cell of any of the aboveembodiments. While few cell therapies and no cell-based gene therapiesare currently approved by the FDA, any of the transgenic techniquesreported herein can be used to prepare a composition to administer to apatient with cancer. Further, CAR-mediated ex vivo expansion can be usedto generate a therapeutically effective amount of a T cell of anisolated T cell of any of the above embodiments.

The processed T cells of the invention can be generated by introducing alentiviral vector containing any of the above-described nucleic acidconstructs into T cells, such as autologous T cells of a patient to betreated for cancer or an IgE-mediated allergic disease. A compositioncomprising autologous T cells is collected from a patient in need ofsuch treatment. The cells are engineered into the processed T cells exvivo, activated and expanded using the methods described herein andknown in the art, and then infused back into the patient. The processedT cells replicate in vivo resulting in persistent immunity againstcancer cells or other cells expressing mIgE.

Any of the above isolated T cells may be processed, with the processed Tcells then transduced with lentiviral vectors as described above togenerate processed T cells for administration. Transduction is carriedout according to known protocols.

The processed T cells are administered to a subject in need of treatmentfor an IgE-mediated allergic disease. The processed T cells are able toreplicate in vivo, providing long-term persistence that can lead tosustained allergic disease control. The processed T cells may beadministered either alone, or as a pharmaceutical composition incombination with one or more pharmaceutically acceptable carriers,diluents or excipients and/or with other components, such as cytokinesor other cell populations. Such compositions may comprise buffers suchas neutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions are preferably formulatedfor intravenous administration. Preferably, the T cells compriseautologous T cells that are removed from the subject and engineered exvivo to express the CAR and administered to the subject.

The processed T cells or pharmaceutical composition thereof may beadministered by a route that results in the effective delivery of aneffective amount of cells to the patient for pharmacological effect.Administration is typically parenteral. Intravenous administration isthe preferred route, using infusion techniques that are commonly knownin immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The quantity of CAR⁺ T cells and frequency ofadministration are determined by such factors as the condition of thepatient, and the type and severity of the patient's disease, althoughappropriate dosages may be determined by clinical trials. An “effectiveamount” is determined by a physician with consideration of individualdifferences in age, weight, disease state, and disease severity of thepatient. Generally, the amount of CAR⁺ T given in a single dosage willrange from about 10⁶ to 10⁹ cells/kg body weight, including all integervalues within those ranges. The CAR⁺ T may be administered multipletimes at these dosages. The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In yet another aspect is provided a method for selecting for a T cellexpressing a transgene of interest, as shown in any of the FIGS. or asdescribed in the description.

In yet another aspect is provided a T cell, as shown in any of the FIGS.or as described in the description.

In another aspect is a method for selectively propagating primary humanT cells resistant to one or more of MTX, 5-FU, Raltitrexed andPemetrexed, as shown in any of the FIGS. or as described in thedescription.

Another aspect is a method of enriching for regulatory T cells in apopulation of T cells isolated from a mammal by contacting saidpopulation with a thymidine synthesis inhibitor selected from the groupconsisting of MTX, 5-FU, Raltitrexed and Pemetrexed, or a combinationthereof, to selectively deplete effector T cells in the population. Insome embodiments, the population of T cells isolated from a mammal iscontacted with both MTX and 5-FU. In some embodiments, the T cellsexpress one or more of DHFR^(FS) and TYMS^(SS). In some embodiments, theT cells express both DHFR^(FS) and TYMS^(SS).

Specific inhibition of 5-aminoimidazole-4-carboxamide riboside (AICAR)synthesis has been shown herein to be neither toxic to T cells norselective for T_(CD4, FoxP3). FoxP3 expression in T_(CD4, FoxP3) has nowbeen found to be enhanced by the specific action of AICARtf inhibition,suggesting some action of AMPK may improve T_(reg) phenotype. Withoutwishing to be bound by theory, isolated T_(reg) studies described hereinshow that the action of MTX is twofold: 1) Selection of T_(reg) isdependent on the depletion of T_(eff), as removal of T_(eff) preventsthe selective increase of T_(reg) following MTX treatment. 2) The actionof MTX does enhance T_(reg) functional activity in some regard aslatency associated peptide (LAP) expression and suppression of T_(eff)proliferation were increased above untreated T_(reg). The activation ofAMPK in the absence of folate depletion by MTX was achieved in thetransgenic T cell experiments and increased the percent of T cells witha functional T_(reg) phenotype. Thus, MTX depletes T_(eff) and promotesan immunosuppressive T_(reg) phenotype.

Another aspect is a method for depleting regulatory T cells in apopulation of T cells isolated from a mammal by culturing saidpopulation in the presence of one or more aminoglycosidases toselectively deplete the regulatory T cells in said culture. In someembodiments, the T cells express one or more of DHFR^(FS) and TYMS^(SS).In some embodiments, the T cells express both DHFR^(FS) and TYMS^(SS).In some embodiments, Treg can be rescuded from G418-mediated depletionwhen Neomycin resistance gene, which prevents G418 toxicity, waspresent. The aminoglycoside depletion may be specifically limited toregulatory T cells. While aminoglycosides have been in use for severaldecades the capacity of this drug to deplete Treg has not beendescribed. Without wishing to be bound by theory, the most likelyexplanation is that the drug is used at much lower doses in vivo thanthose used to deplete Treg in vitro, and is often discontinued fortoxicity to multiple tissues.

In some embodiments, aminoglycosides can be administered to a patientwith a tumor in order to enhance anti-tumor activity. Aminoglycosidescan be administered by pretreatment in a therapy, for example.

Another aspect is a method for selecting for a regulatory T cellisolated from a mammal. The method comprises treating a plurality of Tcells expressing one or more of DHFR^(FS) and TYMS^(SS) with a thymidinesynthesis inhibitor and selecting a regulatory T cell that expresses amarker for a regulatory T cell. In some embodiments, the T cells expressDHFR^(FS). In some embodiments, the selecting step comprises cellisolating with magnetic bead sorting using one or more of an anti-CD4antibody, an anti-CD25 antibody, an anti-CD3 antibody, an anti-CD8antibody, an anti-CD25 antibody, an anti-CD39 antibody, an anti-CD45antibody, an anti-CD152 antibody, an anti-KI-67 antibody, and ananti-FoxP3 antibody. In some embodiments, the thymidine synthesisinhibitor is selected from the group consisting of methotrexate (MTX),5-FU, Raltitrexed or Pemetrexed. In some embodiments, the method furthercomprises treating the regulatory T cell with one or more of folate,leucovarin and FU.

As further demonstrated herein, AThyRs protect AThyRs T cells fromanti-folate toxicity from MTX or Pem. Results described herein establishthat MTX is more toxic to T cells than Pem and that MTX susceptibilityto <1 μM MTX could be completely abrogated by the codon optimization ofDHFR^(FS) or by the addition of TYMS^(SS) to DHFR^(FS) in T cells.Concentrations of up to 1 μM MTX are achieved during the treatment ofrheumatoid arthritis. Higher doses of MTX are achieved in cancerchemotherapy (>1 mM MTX) with the use of leucovorin. Leucovorin rescuesthymidine synthesis through the same pathway as combination DHFR^(FS)and TYMS^(SS). Thus, it is believed that [DHFR^(FS) & TYMS^(SS)]⁺ Tcells will likely resist cytotoxicity induced by the range of MTXexperienced for both immune suppression and cancer treatment.

In another aspect is provided a composition comprising a first pluralityof T cells isolated from a mammal and a thymidine synthesis inhibitor.The first plurality of T cells is enriched for regulatory T cells ascompared to a second plurality of T cells isolated from a mammal thatdoes not comprise a thymidine synthesis inhibitor.

In various embodiments of any of the above aspects and embodiments, Tcells (T lymphocytes) as used herein may comprise or consist of anynaturally occurring or artificially (e.g., synthetically, genetically,recombinantly) engineered immune cells expressing naturally occurring ormade to express or present on the cell surface artificially (e.g.,synthetically, genetically, recombinantly) engineered T cell receptorsor portions thereof, including, for example but not limited to,chimeric, humanized, heterologous, xenogenic, allogenic, and autologousT cell receptors.

In various embodiments of any of the above aspects and embodiments, “Tcells” as used herein include all forms of T cells, for example, but notlimited to T helper cells (T_(H) cells), cytotoxic T cells (T_(c) cellsor CTLs), memory T cells (T_(CM) cells), effector T cells (TEM cells),regulatory T cells (Treg cells; also known as suppressor T cells),natural killer T cells (NKT cells), mucosal associated invariant Tcells, alpha-beta T cells (Tαβ cells), and gamma-delta T cells (Tγδcells).

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention as claimed. The accompanyingdrawings, which are incorporated herein by reference, and whichconstitute a part of this specification, illustrate certain embodimentsof the invention and together with the detailed description, serve toexplain the principles of the present invention.

All cited patents and publications referred to in this application areherein incorporated by reference in their entirety.

Example 1 Materials and Methods Cells and Culture Conditions:

Cells: Peripheral blood mononuclear cells (PBMC) derived from healthydonors at the Gulf Coast Regional Blood Bank or MDACC Blood Bank, bothin Houston, Tex., was subjected to density gradient centrifugation usingFicoll-Paque Plus (GE Healthcare Biosciences, Piscataway Township, N.J.;Cat No. 17-1440-02). PBMC were washed once in CliniMACS Plus PBS/EDTAbuffer (Miltenyi Biotec, Gladbach, Germany, Cat. No. 130-070-525) andtwice in Dulbecco's PBS (D-PBS) (Sigma-Aldrich, St. Louis, Mo., Cat. No.D8537) before resting in complete media (CM) made of RPMI 1640 (ThermoScientific Hyclone, Bridgewater, N.J.; Cat. No. SH30096.01), 10%heat-inactivated fetal bovine serum (FBS—Thermo Scientific Hyclone, Cat.No. SH30070.03), and 2 mM GlutaMAX supplement (Life Technologies, GrandIsland, N.Y.; Cat. No. 35050061). Alternatively, PBMC were frozen usinga prepared mixture of 50% heat-inactivated FBS, 40% RPMI 1640, and 10%DMSO (Sigma-Aldrich, PA; Cat. No. D2650)—freeze media (FM) at 4×107cells/mL. The use of rested or frozen PBMC is outlined in eachexperiment, below. The Jurkat cell line, a human T cell acutelymphoblastic leukemia (American Type Culture Collection, Manassas, Va.,Cat. No. TIB-152) was used and maintained in CM. The identity of thiscell line was assured by short tandem repeat DNA fingerprintingperformed by MDACC Cancer Center Support Grant Characterized Cell LineCore. Activating and propagating cells (AaPC) were used to stimulate Tcells. The AaPC cell line K562 clone.4, expressing CD86, CD137, CD64,along with membrane bound IL-15, was modified to present OKT3 antibodyfor the polyclonal stimulation of T cells, as previously described(Singh et al., Journal of immunotherapy 2014, 37(4):204-213). For thepropagation of chimeric antigen receptor (CAR)+ T cells, the AaPCCARL+K562 (Rushworth et al., Journal of immunotherapy 2014,37(4):204-213) was utilized.

All AaPC were rapidly thawed in a 37° C. water bath and washed twicebefore stimulation of T cells (Singh et al., supra). Jurkat and AaPCwere tested for the presence of mycoplasma before use. Cell counting wasaccomplished in a mixture of 0.1% Trypan Blue (Sigma-Aldrich, T8154)with the Cellometer K2 Image Cyotmeter (Nexcelom, Lawrence, Mass.).

Chemical and Biological Agents:

Stimulation via CD3 and CD28 was achieved by the addition of 30 ng/mLOKT3 antibody (eBioscience, San Diego, Calif., Cat. No. 16-0037-85), 100ng/mL anti-CD28 antibody (EMD Millipore, Temecula, Calif., Cat. No.CBL517). T cell stimulation included recombinant human IL-2 (Proleukin,Prometheus Labs, San Diego, Calif.). When indicated, the following drugswere used: 5-FU, MTX, pemetrexed, raltitrexed, G418, and AP20187.Further information regarding each drug is given in Table 1.

DNA Expression Plasmids:

DNA plasmids for testing anti-thymidylate resistance (AThyR) transgeneswere generated using the previously described DNA plasmid G4CAR as abackbone (Rushworth et al., supra). Commercially synthesizedFLAG-DHFR^(FS), codon optimized (CoOp) DHFR^(FS), FLAG-TYMS^(SS), andCoOp TYMS^(SS) DNA (Life Technologies, Gene Art), and neomycinresistance gene (NeoR) DNA product were cleaved by NheI and ApaI.Reporter genes mCherry with N-terminus SV40 nuclear localizationsequence (RFP), inducible suicide gene CoOp iC9 (both produced byGeneArt), and enhanced green fluorescent protein.

TABLE 1 Chemical Agents Agent Manufacturer ID No. 5-fluorouracil APPPharmaceuticals, Schaumburg, IL NDC 63323-117-10 Methotrexate Hospira,Lake Forest IL NDC 61703-350-38 Pemetrexed Lilly, Indianapolis, IN NDC0002-7610-01 Raltitrexed Abcam Biochemicals, Cambridge, MA AB142974 G418Invivogen, San Diego, CA Ant-gn-1 AP20187 Clontech, Mountain View, CA635060

(eGFP) DNA were digested by ApaI and KpnI. The G4CAR backbone wasrestriction enzyme digested by NheI and KpnI. The G4CAR backbone wasligated with NheI and ApaI digested fragments and ApaI and KpnI digestedfragments in a three component ligation. Enzyme digestion locations ofNheI, KpnI, and ApaI are shown in FIG. 1B. The drug resistant component(DHFR^(FS), TYMS^(SS), or NeoR) was permutated with the transgenes (RFP,CoOp iC9, and GFP) to make the following DNA plasmids:FLAG-DHFR^(FS)-2A-eGFP pSBSO (DG), FLAG-CoOp DHFR^(FS)-2A-eGFP pSBSO(CoOp DG); FLAG-TYMS^(SS)-2A-GFP pSBSO (TSG); FLAG-CoOp TYMS^(SS)-2A-GFPpSBSO (CoOp TSG); FLAG-TYMS^(SS)-2A-RFP pSBSO (TSR); NeoR-2A-GFP pSBSO(NRG); and FLAG-DHFR^(FS)-2A-iC9 pSBSO (DFSiC9). The constructFLAG-TYMS^(SS)-2A-IL-12p35-2A-IL-12p40 pSBSO (TS^(SS)IL-12) wassynthesized from codon optimized (GeneArt, Life Technologies) IL-12 p35and IL-12 p40 transgenes and digested within the 2A regions to ligateIL-12 p35 and IL-12 p40 with a TYMS^(SS) fragment also digested withinthe 2A region. TS^(SS)G backbone digestion points 5′ to the start siteof TYMS^(SS) and 3′ to the IL-12p40 stop site ligated the threecomponents into the TS^(SS)G backbone in a four part ligation. Aconstruct is also provided, which encodes Myc-DHFR^(FS)-2A (thepolypeptide sequence corresponding to Myc-DHFR^(FS)-2A is provided asSEQ ID NO: 10). The polypeptide sequence for TYMS^(SS) is provided asSEQ ID NO: 11. The polypeptide sequence for DHFR^(FS) is provided as SEQID NO: 12. Codon optimization of DHFR^(FS) and TYMS^(SS) DNA wasperformed to avoid the mRNA transcript from being bound by DHFR and TYMSproteins, respectively. Known RNA binding motifs of DHFR and TYMS mRNAare recognized by DHFR (Tai et al., The Biochemical journal 2004, 378(Pt3):999-1006) and TYMS (Lin et al., Nucleic acids research 2000,28(6):1381-1389), respectively. Codons of DHFR^(FS) and TYMS^(SS) werealtered as much as possible while maintaining the amino acid sequence ofeach protein in order to avoid protein binding of the mRNA transcript.Previously described CD19-specific chimeric antigen receptor (CAR)(Rushworth et al., supra) was utilized without modification.

Myc-ffLuc-NeoR pSBSO (NRF) was constructed using the backbone ofCD19-2A-Neo pSBSO (Rushworth et al., supra) isolated after restrictiondigestion with NheI and SpeI. NheI and SpeI digested Myc-fireflyLuciferase (ffLuc) insert was ligated to CD19-2A-Neo backbone followedby digestion of the ligation product with SpeI and EcoRV. SpeI and EcoRVdigested NeoR fragments were then ligated to the digested backbone toyield NRF. All constructs contain Sleeping Beauty (SB) indirect/directrepeat (IR/DR) sites to induce genomic integration in the presence of SBtransposase. Each transgene is promoted using elongation factor 1 alpha(EF1-α) promoter. Cartoon representations of constructs can be seen inFIG. 1 B and FIG. 8A. Select DNA and protein sequences can be found inTable 2.

TABLE 2 Synthetic DNA/protein sequences FLAG-AtggactacaaggacgacgacgacaaggattacaaggatgatgatgataaggactataaagacgacgatgatadmDHFRaggacgtcgttggttcgctaaactgcatcgtcgctgtgteccagaacatgggcatcggcaagaacggggacttcccctggccaccgctcaggaatgaatccagatatttccagagaatgaccacaacctettcagtagaaggtaaacagaatctggtgattatgggtaagaagacctggttctccattectgagaagaatcgacctttaaagggtagaattaatttagttctcagcagagaactcaaggaacctccacaaggagctcattttctttccagaagtctagatgatgccttaaaacttactgaacaaccagaattagcaaataaagtagacatggtctggatagttggtggcagttctgtttataaggaagccatgaatcacccaggccatcttaaactatttgtgacaaggatcatgcaagactttgaaagtgacacgifitttccagaaattgatttggagaaatataaacttctgccagaatacccaggtgttctctctgatgtccaggaggagaaaggcattaagtacaaatttgaagtatatgagaagaatgat (SEQ ID NO: 1) FLAG-CoOpAtggactacaaggacgacgacgacaaggattacaaggatgatgatgataaggactataaggacgatgatgacadmDHFRaagacgtcgtgggcagcctgaactgcatcgtggccgtgtcccagaacatgggcatcggcaagaacggcgacttcccctggccccctctgcggaacgagagccggtacttccagcggatgaccaccaccagcagcgtggaaggcaagcagaacctcgtgatcatgggcaagaaaacctggttcagcatccccgagaagaaccggcccctgaagggccggatcaacctggtgctgagcagagagctgaaagagccccctcagggcgcccacttcctgagcagatctctggacgacgccctgaagctgaccgagcagccagagctggccaacaaggtggacatggtgtggatcgtgggcggcagctccgtgtacaaagaagccatgaaccaccctggccacctgaaactgttcgttacccgtataatgcaggatttcgagagcgataccttettccccgagatcgacctggaaaagtacaagctgcttcccgagtaccccggcgtgctgtccgatgtgcaggaagagaagggcatcaagtacaagttcgaggtgtacgagaagaatgac (SEQ ID NO: 2) FLAG-AtgtatccgtacgacgtaccagactacgcatatccgtacgacgtaccagactacgcagacgtccctgtggccggdmTYMSctcggagctgccgcgccggcccttgccccccgccgcacaggagcgggacgccgagccgcgtccgccgcacggggagctgcagtacctggggcagatccaacacatcctccgctgcggcgtcaggaaggacgaccgctcgagcaccggcaccctgtcggtattcggcatgcaggcgcgctacagcctgagagatgaattccctctgctgacaaccaaacgtgtgttctggaagggtgttttggaggagttgctgtggtttatcaagggatccacaaatgctaaagagctgtcttccaagggagtgaaaatctgggatgccaatggatcccgagactttttggacagcctgggattctccaccagagaagaaggggacttgggaccagtttatggcttccagtggaggcattttggggcagaatacagagatatggaatcagattattcaggacagggagttgaccaactgcaaagagtgattgacaccatcaaaaccaaccctgacgacagaagaatcatcatgtgcgcttggaatccaagagatcttcctctgatggcgctgcctccatgccatgccctctgccagttctatgtggtgaacagtgagctgtcctgccagctgtaccagagatcgggagacatgggcctcggtgtgcctttcaacatcgccagctacgccctgctcacgtacatgattgcgcacatcacgggcctgaagccaggtgactttatacacactttgggagatgcacatatttacctgaatcacatcgagccactgaaaattcagcttcagcgagaacccagacctttcccaaagctcaggattcttcgaaaagttgagaaaattgatgacttcaaagctgaagactttcagattgaagggtacaatccgcatccaactattaaaatggaaatggctgtt (SEQ ID NO: 3) FLAG-CoOp-AtggactacaaggacgacgacgacaaggattacaaggatgatgatgataaggactataaggacgatgatgacadmTYMSaagacgtccccgtggccggcagcgagctgcctagaaggcctctgcctcctgccgctcaggaaagggacgccgaacctagacctcctcacggcgagctgcagtacctgggccagatccagcacatcctgagatgcggcgtgcggaaggacgacagaagcagcacaggcaccctgagcgtgttcggaatgcaggccagatacagcctgcgggacgagttccctctgctgaccaccaagegggtgttctggaagggcgtgctggaagaactgctgtggttcatcaagggcagcaccaacgccaaagagctgagcagcaagggcgtgaagatctgggacgccaacggcagcagagacttcctggacagcctgggcttcagcaccagagaggaaggcgatctgggtcccgtgtacgggtttcaatggcggcacttcggcgccgagtatcgggacatggagagcgactacagcggccagggcgtggaccagctgcagagagtgatcgacaccatcaagaccaaccccgacgaccggcggatcatcatgtgcgcctggaaccccagagatctgcccctgatggccctgcctccatgtcacgccctgtgccagttctacgtcgtgaactccgagctgagctgccagctgtaccagcggagcggcgatatgggactgggcgtgcccttcaatatcgccagctacgccctgctgacctacatgatcgcccacatcaccggcctgaagcccggcgactttatccacaccctgggcgacgcccatatctacctgaaccacatcgagcccctgaagattcagctgcagcgcgagcccagacccttcccaaagctgcggatcctgcggaaggtggaaaagatcgacgacttcaaggccgaggacttccagatcgagggctacaacccccaccccacaatcaagatggaaatggccgtg (SEQ IDNO: 4) eGFP forward5′ cccgggcccggcgccatgccacctcctcgcctcctcttc 3′ (SEQ ID NO: 5)eGFP reverse5′ ggtacccttgtacagctcgtccatgccgagagtgatcccggcggcggtcac 3′ (SEQ ID NO: 6)NeoR forward5′ gctagcacatgtgccaccatgattgaacaagatggattgcacgcaggttctccggccgcttgg 3′ (SEQID NO: 7) Neo R reverse5′ aagcttccgcggccctctccgctaccgaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatc3′ (SEQ ID NO: 8) NLS MAPKKKRKVGIHRGVP (SEQ ID NO: 9)

Genetic Modification and Propagation of Cells:

The Amaxa Nucleofector® II (Lonza, Allendale, N.J.) was used toelectroporate both Jurkat and human PBMC. Electroporation of Jurkatcells utilized a modified buffer (Chicaybam et al., Proceedings of theNational Academy of Sciences of the United States of America 2002,99(6):3400-3405) containing 5 mM KCl, 15 mM MgCl₂, 120 mMNa₂HPO₄/NaH₂PO₄, pH 7.2, and 50 mM DMSO, where 10⁶ Jurkat cells percuvette were electroporated using program T-14 before immediate transferto CM. The addition of drug occurred 48 hours after electroporation andcell culture remained undisturbed until sampling for gene expression ondays 10-12 post electroporation. Human PBMC electroporation followed apreviously described protocol (Rushworth et al., supra). Briefly, 1 to2×10⁷ thawed PBMC per cuvette were electroporated in Amaxa T cellNucleofector solution (Lonza Biosciences; Cat No. VPA-1002) usingprogram U14. On the following day, PBMC were stimulated in fresh CM withAaPC at a ratio of 1:1 including 50 IU/mL IL-2, unless otherwise noted.The cellular co-culture concentration of 10⁶ cells/mL was maintained ateach stimulation, and PBMC derived T cells were re-stimulated every 7days using the same concentrations. IL-2 was added when media waschanged between stimulations. Drug treatment initiated 48 hours afterco-culture began and continued until day 14. Drug was only added withfresh CM.

Western Blot:

10⁶ T cells were centrifuged from culture, supernatant aspirated, andthe pellet rapidly frozen in liquid nitrogen. Whole-cell extracts wereharvested using 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5%deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 150 mM p-nitrophenylphosphate and 0.3 μM Aprotinin, pH 7.4. Proteins were separated bySDS-PAGE in reducing conditions and analyzed using specific primaryantibodies indicated in Table 3. Detection was performed using anenhanced chemiluminescence detection system.

Flow Cytometry:

Cultured cells were resuspended, and washed once in FACS stainingsolution (Rushworth et al., supra). If transgene expression alone wassought, the specimen was then analyzed on a flow cytometer. The BDLSRFortessa (BD Biosciences) was used to analyze RFP expression;otherwise, BD FACSCalibur (BD Biosciences) was used. Surface antibodystaining was performed in FACS staining solution withfluorochrome-conjugated antibodies at 4° C. for at least 30 minutes.Antibody targets, concentrations, and manufacturers are listed in Table4. Analysis of flow cytometry data utilized FlowJo v 10.0.5 (Tree StarInc., Ashland, Oreg.).

Luciferase Assay:

Cultured T cells were tested for the persistence of ffLuc transgene bythe cleavage of D-luciferin (Perkin Elmer, Waltham, Mass., Cat. No.122796). Resuspended cells were plated and washed once in D-PBS beforetesting in a D-PBS solution of D-luciferin at 0.14 mg/mL. Afterincubation at 37° C. for 10 min, the plate was analyzed on a TopCountNXT Luminescence Counter (Perkin Elmer).

TABLE 3 Western Blot Antibodies Antibody Manufacturer Cat. No. DilutionActin Sigma A2228 1:10000 Hsp-70 Santa Cruz Biotechnology, SC-24 1:5000Dallas, TX DHFR Santa Cruz Biotechnology SC-377091 1:1000 TYMS MilliporeMAB4130 1:1000 Myc Tag CST 2276S 1:1000 DYKDDDDK Tag Pierce MA1-918761:1000

TABLE 4 Flow Cytometry Antibodies Antibody Manufacturer Cat. No.Dilution CD3-APC BD Pharmingen 340661 1:33 CD3-PerCP-Cy5.5 BD Pharmingen340949 1:33 CD4 FITC BD Pharmingen 340133 1:33 CD4-PE BD Pharmingen347327 1:33 CD4-PerCP-Cy5.5 BD Pharmingen 341645 1:33 CD8-APC BDPharmingen 340359 1:33 Annexin V-PE BD Pharmingen 556422 1:20 7-AAD BDPharmingen 559925 1:20 Propidium Iodide BD Pharmingen 556463 Human Fc-PEInvitrogen H10104 1:40 Myc- AF488 MBL M047-A48 1:33 FLAG-AF647 CellSignaling 3916S 1:33

Chromium Release Assay:

Antigen specific cytotoxicity was assessed by CRA. This assay waspreviously described (Rushworth et al., supra). Briefly, antigenpositive CD19+EL-4 were compared to antigen negative CD19^(neg) EL-4after each cell line was loaded with ⁵¹Cr for 3 hours and subsequentlyincubated with CD19-specific CAR+ T cells at a 1 target:5 effector cellratio for 6 hours. Release of ⁵¹Cr from cell lysis was assessed by theTopCount NXT scintillation counter.

Statistical Analysis:

Statistical analysis and graphical representation of data was achievedusing Prism v6.0 (Graph Pad Software Inc., La Jolla, Ca). Experiments ofmore than one variable were analyzed by multivariate analysis: Two-WayANOVA was used when appropriate with Sidak's multiple comparison test,One-Way ANOVA was used when appropriate with Tukey's or Dunnett'smultiple comparison tests as applicable, non-Gaussian distributions wereassessed by the Kruskall-Wallis test followed by Dunn's multiplecomparison test. Single variable tests (experimental vs. control) weremade using the Mann-Whitney test. Statistical significance wasdesignated as α<0.05.

Results A. Testing AThyR Transgene Selection in Jurkats

DHFR^(FS) were used to determine whether T cells can begenetically-modified to resist toxic doses of AThys used in the initialtreatment of malignancy. DHFR^(FS+) T cells resistant to MTX aredescribed by Jonnalagadda et al., PloS one 2013, 8(6):e65519, andJonnalagadda et al., Gene therapy 2013, 20(8):853-860. 5-FU resistantTYMS muteins previously identified within a bacterial culture system(Landis et al., Cancer research 2001, 61(2):666-672) were tested inhuman cells (data not shown) and TYMS^(SS) was chosen for further study.

To test the enhanced survival of each AThyR, constructs individuallyexpressing DHFR^(FS), TYMS^(SS), and NeoR were ligated into the samebackbone containing Sleeping Beauty (SB) transposable elements upstreamof eGFP (FIG. 1B). eGFP was used to track the predominance of survivinggenetically-modified T cells. Jurkat cells were co-electroporated witheach construct and SB11 transposase (Singhet et al., Cancer Research2008, 68(8):2961-2971), which mediated genomic integration of eachconstruct. Cytotoxic drugs were added two days after electroporation.Jurkat were assessed for eGFP expression in viable cells by propidiumiodide (PI) exclusion on day 10-12 (FIG. 1C). Increased percentageexpression of eGFP was sought as a measure for transgene selection inthe presence of drug. Overall survival and mean fluorescence intensity(MFI) of eGFP are also given in FIGS. 2AI and AII, respectively.Overall, the data demonstrate that DHFR^(FS) has much better selectionthan the traditional drug-resistance transgene NeoR. The data alsodemonstrate that TYMS^(SS) has no independent capacity to enhance Jurkatsurvival.

More specifically, it was found that DHFR^(FS) confers resistance to MTXat concentrations range of 0.01-0.5 μM, and codon optimization ofDHFR^(FS) enhanced the drug resistance range of CoOp DHFR^(FS) to 0.01-1μM (FIG. 1C). Codon optimization removed potential endogenous DHFRbinding to the DHFR^(FS) mRNA as well as possible micro RNA bindingdomains. Notably, gating on eGFP⁺ cells demonstrated that DHFR^(FS)constructs lead to a MTX dependent increase in eGFP MFI. Hence, eGFPexpression within a single cell increased based on the addition of MTX.This finding occurred independent of mRNA regulation until 5 μM MTXwhere endogenous codon DHFR^(FS) expression significantly decreasedcompared to CoOp DHFR^(FS) (p<0.0001) (FIG. 2A-II). Drug inducibletransgene expression is a rare phenomenon. This phenomenon, althoughrare, is not novel. While the capacity of DHFR to increase cis-expressedeGFP in an MTX dependent manner was previously described for nativeDHFR, the phenomenon was attributed to MTX binding DHFR, DHFR releasingDHFR mRNA, and free DHFR mRNA leading to increased translation of DHFRprotein (Meyer et al., Proceedings of the National Academy of Sciencesof the United States of America 2002, 99(6):3400-3405). Here it is notedthat the phenomenon also occurs with MTX resistant DHFR^(FS), and withDHFR^(FS) occurs independent of mRNA regulation from 0.01-1 μM MTX.Hence, without wishing to be bound by any theory, it is believed thatthe regulation of DHFR expression occurs partially through a heretoforeunknown mRNA independent mechanism.

There was no drug selective advantage for TYMS^(SS) expressing Jurkatwhen tested with 5-FU (FIG. 1C). Native codon TYMS^(SS) had noexpression advantage over No DNA Jurkat at any concentration of 5-FU.Further analysis of eGFP⁺ cells for eGFP MFI revealed that TYMS^(SS)expressed at a lower eGFP MFI compared to CoOp TYMS^(SS) (FIG. 2A). Itis concluded that lower expression of TYMS^(FS) due to mRNA basedsuppression contributed to the lack of TYMS^(SS) survival advantage.When mRNA regulatory mechanisms are ablated by codon optimization,TYMS^(SS) has a significant expression advantage over mockelectroporated Jurkat, and a weak survival advantage in 5 μM 5-FU.Without wishing to be bound by any theory, the lack of significantlyenhanced survival is likely due to an alternative mechanism of 5-FUcontributing to toxicity.

NeoR was used to select for enhanced survival of Jurkat in the presenceof G418. This was intended to serve as a standard to gauge the utilityof DHFR^(FS) and TYMS^(SS). Electroporation of NeoR into Jurkat improvedsurvival in the presence of G418 at 0.72-1.1 mM G418 (FIG. 1C). Thesurvival advantage of NeoR over No DNA was not significant due tovariability (FIG. 2A), but a G418 dependent increase in GFP MFI wasnoted. The GFP MFI significantly increased above No DNA Jurkat at 1.4 mMG418 (FIG. 2A-II). These results reinforce that DHFR^(FS) and NeoR arecapable of providing dose-dependent transgene selection advantage insurviving Jurkat. However, only DHFR^(FS) conferred reliable survivaladvantages to Jurkat in this experiment (FIG. 2A-II).

The next experiment combined DHFR^(FS) and TYMS^(SS) byco-electroporating each plasmid into Jurkat. The capacity of thecombined transgenes to resist commonly used anti-folate AThys: MTX, Pem,and Raltitrexed (Ral), were tested. As before, drug was added on day 2and cells were tested on day 10-12. There was clear selection for[DHFR^(FS) & TYMS^(SS)] expressing Jurkat in 0.1-1 μM MTX when comparedto similarly treated No DNA or untreated [DHFR^(FS) & TYMS^(SS)]⁺ Jurkat(FIG. 1D). It should be noted that endogenous codon DHFR^(FS) was usedin these experiments and the resistance to MTX was enhanced from 0.5(FIG. 1C) to 1 μM MTX (FIG. 1D) by the addition of TYMS^(SS) with noother changes to the experimental conditions. Selection was also notedfor 50-100 μM Pem (FIG. 1D). Moderate selection was also noted with 10μM Ral when compared to untreated [DHFR^(FS) & TYMS^(SS)]⁺ Jurkat (FIG.2B). Ral primarily targets TYMS, whereas MTX and Pem target both DHFRand TYMS (Walling, Investigational new drugs 2006, 24(1):37-77), hencethe improved selection for MTX and Pem over Ral in [DHFR^(FS) &TYMS^(SS)]⁺ Jurkat. After 2 weeks within 1 μM MTX, surviving [DHFR^(FS)& TYMS^(SS)]⁺ Jurkat were refreshed in untreated media and grown for 3-5weeks. Subsequently, the stability of transgene expression of [DHFR^(FS)& TYMS^(SS)]+ Jurkat was tested by flow cytometry with the co-expressionof eGFP representing DHFR^(FS) expression and RFP representing TYMS^(SS)expression as seen in FIG. 1E. Each color represents a separateexperiment and is overlaid to represent the trend that DHFR^(FS) andTYMS^(SS) co-express in a correlated fashion. In fact, analysis of GFPMFI representing DHFR^(FS) expression and RFP MFIs representingTYMS^(SS) expression over multiple anti-folate drugs, at multipleconcentrations demonstrated that DHFR^(FS) & TYMS^(SS) co-express with astrong Pearson's correlation (R²=0.9) (FIG. 2C). Without wishing to bebound by theory, this finding suggests that expression of DHFR^(FS) issomehow regulated by the expression of TYMS^(SS), or vice versa.

B. Selective Propagation of Primary Human T Cells Resistant to MTXand/or 5-FU.

As demonstrated, TYMS^(SS) enhances the ability of Jurkat expressingDHFR^(FS) to survive in the presence of MTX and Pem, which both targetendogenous DHFR and TYMS to prevent thymidine synthesis. Given the morerobust survival to toxic MTX concentrations conferred by DHFR^(FS) andTYMS^(SS), experiments with MTX were undertaken to demonstrateanti-folate and AThy resistance. TYMS^(SS) with DHFR^(FS) were tested inhuman cells by electroporation into human PBMC. The day followingelectroporation, cells were stimulated with an OKT3-loaded AaPC capableof polyclonal T cell propagation. The propagation schematic is shown inFIG. 3A. Two days after AaPC stimulation, the co-cultures received 0.1μM MTX, 5 μM 5-FU, or 1.4 mM G418 until day 14, as designated in FIG. 3.The co-cultures were re-stimulated with AaPC at a 1:1 ratio and given 50IU/mL IL-2 every 7 days from day 1 to 35. Phenotypic changes intransgene expression were tracked during drug administration for thefirst 14 days and for the 21 days after drug administration had ended.The weekly changes in transgene expression can be noted in FIG. 3B-I,C-I, D-I.

Initial testing of DHFR^(FS), TYMS^(SS), and NeoR co-expressed withfluorescent proteins demonstrated rapid and persistent selection tonearly complete selection for expression of DHFR^(FS) with MTX and NeoRwith G418 (FIG. 3B-I). Survival and propagation of AThyR+ T cells(TAThyR) compared to No DNA T cells on day 21 showed that the presenceof AThyR or NeoR transgene plays a role in T cell survival and growth(FIG. 4A). On day 35, total inferred cell count for T cells expressingAThyR and NeoR transgenes were compared to untreated No DNA T cells, andNeoR⁺ T cells were the only T cells with significantly inferior growthat Day35 (FIG. 4B-I). In opposition to experiments in Jurkat, TYMS^(SS)demonstrated selection within the population of surviving T cells on Day21 in the presence of 5-FU. However, the selected TYMS^(SS) expressing Tcells did not persist to Day 35, and the lack of persistence was alsonoted when [DHFR^(FS) & TYMS^(SS)] were selected using MTX and 5-FU.Without wishing to be bound by any theory, thymidine synthesis may berestored by TYMS^(SS) and thymidine transporters then make thymineavailable to un-transformed cells. Without wishing to be bound by anytheory, this is likely mediated by an equilibrative nucleosidetransporter as the same transporter that permits 5-FU entry alsomediates equilibrative transport of thymine. As TYMS^(SS) restoresthymidine synthesis in the presence of methotrexate, DHFR^(FS) is nolonger able to select for T cells expressing DHFR^(FS) & TYMS^(SS) asnoted in FIG. 3B-I.

In order to achieve complete selection of TYMS^(SS) for possible use incombination therapies, NeoR was co-electroporated into primary T cellswith DHFR^(FS), TYMS^(SS), and [DHFR^(FS) & TYMS^(SS)]. The only changemade to the propagation method was the addition of 100 IU/mL IL-2 ratherthan 50 IU/mL from days 14-35 to supplement the poor outgrowth alreadynoted in G418 selected T cells. The higher doses of IL-2 wereinsufficient to rescue poor outgrowth when G418 and 5-FU were combinedfor T cell selection (FIG. 4B-II). With the co-transfection of NeoR intoDHFR^(FS) and/or TYMS^(SS) expressing T cells, nearly 100% transgenesselection was observed with the same transgene selection kinetics amongall groups (FIG. 3C-I).

The influence of TYMS^(SS) on DHFR^(FS) selection in T cells subjectedto MTX was tested. Plasmids expressing DHFR^(FS) were co-electroporatedinto T cells along with either TYMS^(SS) co-expressing RFP or a vectorexpressing RFP alone. This experiment followed the same strategy asdescribed for FIG. 3B. Due to technical limitations, the total amount ofDHFR^(FS) expressing plasmid DNA electroporated into the same number ofT cells was decreased. Consequently, fewer T cells initially expressedDHFR^(FS) at the beginning of the experiment and DHFR^(FS) wasincompletely selected by the addition of MTX within a 14 day time period(FIG. 3D-I). The progressive loss of DHFR^(FS) after day 14 isreminiscent of TYMS^(SS) expression in FIG. 3B-I. This demonstrates thatAThyR transgenes must select for a large portion of the T cellpopulation to maintain stable expression within the population. Withregards to the influence of TYMS^(SS) on the selection of DHFR^(FS), itappears that TYMS^(SS) blunts DHFR^(FS) selection in T cells asselection of [DHFR^(FS) & RFP] expressing T cells was more robust thanselection of [DHFR^(FS) & TYMS^(SS)] expressing T cells. This isattributed to the restoration of thymidine synthesis in the presence ofTYMS^(SS) (FIG. 3D-I). The presence of 5-FU prevents selection ofDHFR^(FS) with or without TYMS^(SS), and this is attributed to theTYMS^(SS) independent inhibition of mRNA and rRNA.

It was also noted that transgenic selection tended to increase thepopulation of CD4⁺ T cells by day 35 in all T cell experiments, whichwas not seen with un-modified T cell cultures. This was noted in anyexperiment involving one or more transgenes selected in the presence ofcytotoxic drug (FIG. 3B-II, 3C-II, 3D-II, respective flow plots seen inFIGS. 6A, 6B, and 6C). The experiment in FIG. 3D-II demonstrates that itis not caused by cytotoxic drug, rather, the presence of transgene incombinations with cytotoxic drug leads to CD4⁺ T cell predominance byday 35. The selection towards CD4⁺ T cell predominance was not noted 7days after initial drug selection for AThyR+ T cells (FIG. 4C), which isconsistent with previously published findings using DHFR^(FS) T cells(Jonnalagadda et al., Gene therapy 2013, 20(8):853-860). The longerperiod of follow-up than prior experiments demonstrated a previouslyunknown phenomenon that CD8⁺ T cells are unable to persist for longperiods of time following cytotoxic insult, or are selectively outgrownby CD4⁺ T cells.

C. MTX Increases Cis-Transgene Expression in DHFR^(FS+) T Cells

MTX mediated changes in transgene expression are useful for in vivocontrol of transgene expression in both animals and humans. Thus,according to the present invention, MTX, a clinically available drug, isused to mediate transgene expression either up or down in T cells. Toinvestigate the persistence of this regulation, DHFR^(FS), CoOpDHFR^(FS), and [DHFR^(FS) & TYMS^(SS)] expressed in Jurkat were selectedin 1 μM MTX for 2 weeks and rested for 3-5 weeks before testing MTXmediated regulation of DHFR^(FS) expression. The expression of DHFR^(FS)and codon optimization (CoOp) DHFR^(FS) selected for uniform expressionin Jurkat I cell line is shown in FIG. 5A. CoOp DHFR^(FS) did notcontribute to a significantly higher expression of DHFR^(FS) asindicated by a cis-expressed eGFP, nor did it prevent MTX inducedincreases in transgene expression as noted in FIG. 5B. This wasunexpected. However, the loss of MTX induced increase in DHFR^(FS)expression was noted when TYMS^(SS) was co-expressed with DHFR^(FS) asseen in FIG. 5A and FIG. 5B. The addition of TYMS^(SS) led to aninsignificant reduction in the expression of native DHFR^(FS) in theabsence of MIX, The addition of MTX was unable to induce the sameincrease in DHFR^(FS) expression seen during the sole expression ofeither DHFR^(FS) version. Thus, TYMS^(SS) is playing a role in the MTXinducible increase of DHFR^(FS). In certain experiments, theco-expression of TYMS^(SS) with DHFR^(FS) in Jurkat blunts the MTXinduced increase in eGFP MFI (FIG. 3B-I). Thus, DHFR^(FS) maintainsMTX-inducible expression of cis-transgenes which is dependent on MTXmediated inhibition of TYMS.

Expression of these transgenes in primary cells was next attempted torecapitulate the findings of MTX inducible increases in DHFR^(FS)expression that were prevented by TYMS^(SS). Expression of DHFR^(FS),TYMS^(SS), or [DHFR^(FS) & TYMS^(SS)] was achieved with stability andpurity by selecting from days 2-14 of propagation with the respectivedrugs MTX, 5-fluorouracil (5-FU), and G418 when the selection vectorcontaining neomycin resistance was included. The expression of DHFR^(FS)linked eGFP and TYMS^(SS) linked RFP can be noted in FIG. 5C forDHFR^(FS), TYMS^(SS), or [DHFR^(FS) & TYMS^(SS)]. Again it is noted thatDHFR^(FS) expression is increased in the presence of MTX (FIG. 5D), butthis increase is blunted and no longer significant when TYMS^(SS) isco-expressed with DHFR^(FS), as in Jurkat. Of note, expression ofTYMS^(SS) without DHFR^(FS) was successfully achieved in primary T cellsby selection with 5-FU and a trans neomycin resistance gene selected byG418. When TYMS^(SS) was tested for inducible changes in the presence ofhigh doses of MTX (FIG. 5D), it was found that TYMS^(SS) linked RFPdecreased significantly. The presence of DHFR^(FS) along with TYMS^(SS)in the same treatment conditions prevented this decrease. MTX induced areduction in the expression of TYMS^(SS) that MTX resistant DHFR^(FS)restored. These findings could indicate that TYMS^(SS) is beingrepressed by a lack of 5, 10-methylenetetrahydrofolate (5, 10 CH₂THF).Without being limited by any particular mechanism, it is proposed thatMTX, which leads to a drop in 5, 10 CH₂THF, is causing TYMS protein tobind TYMS and TYMS^(SS) mRNA preventing expression. It should be notedthat TYMS^(SS) is equivalent to the native sequence with the exceptionof the point mutations.

Based on findings in FIG. 5A-B, it is proposed that DHFR^(FS) expressionis also regulated by the synthesis of thymidine. Likewise, based onfindings in FIGS. 5C & D, it is proposed that TYMS^(SS) expression isregulated by the synthesis of tetrahydrofolate (THF). As a derivative ofTHF is used to make thymidine, a logical conclusion was made thatDHFR^(FS) regulates the expression of TYMS^(SS) and TYMS^(SS) regulatesthe expression of DHFR^(FS). Therefore, a correlated expression ofDHFR^(FS) and TYMS^(SS) should be noted within individual cells. When acorrelated expression of DHFR^(FS) and TYMS^(SS) was tested by observingflow plots of Jurkat in FIG. 5E and primary T cells in FIG. 5F, it wasnoted. A control RFP vector co-expressed with DHFR^(FS), but notmodulated by cis expression with TYMS^(SS), did not appear to have thesame co-expression pattern (FIG. 5F). To quantify this observation,Jurkat expressing [DHFR^(FS) & TYMS^(SS)] were treated with antifolatesMTX, pemetrexed, and raltitrexed at varying concentrations for 2 weeks.DHFR^(FS) linked eGFP MFI and TYMS^(SS) linked RFP MFI for each separatewell were then plotted and correlated. The linked expression betweenDHFR^(FS) and TYMS^(SS) was significant and fit a linear regression(FIG. 5G). These findings support a general mechanism for regulation ofDHFR and TYMS, which leads to a linear co-expression of DHFR^(FS) andTYMS^(SS). This model is shown in FIG. 511.

Based on the above model in FIG. 511, it appears that TYMS^(SS)expression will be stabilized by DHFR^(FS) from strong expressionchanges in the presence of MTX. This was tested in FIG. 5I with primaryT cells expressing DHFR^(FS) along with either RFP or TYMS^(SS) linkedto RFP by applying increasing doses of MTX. As expected, DHFR^(FS)linked eGFP was increased by increasing concentrations of MTX, and thisincrease was blunted by the presence of TYMS^(SS) (FIG. 5I-J). Thisconserves the model in FIG. 511 where restoration of thymidine synthesisprevents the MTX induced increase in DHFR^(FS). Further conserving themodel, RFP linked to TYMS^(SS) did not significantly increase over anyconcentration of MTX used (FIG. 5I-J). When DHFR^(FS) linked eGFPincreased so too did the control RFP, and an increase in the expressionof RFP alone was not expected. A possible explanation is that thisincrease was noted above 0.5 μM MTX, and DHFR^(FS) alone is onlyresistant to 0.5 μM MTX.⁹ This suggests that higher doses of MTX beginto select for cells with higher gene content of DHFR^(FS) and associatedtransgenes. Notably, DHFR^(FS) co-expressed with TYMS^(SS) is resistantto concentrations of up to 1 μM MTX. This further supports the use ofTYMS^(SS) to modulate transgene expression and prevent unwantedselection towards higher gene expression levels of genes expressed incis or trans with DHFR^(FS).

Next, a construct of DHFR^(FS) cis expressing an inducible suicidegene—inducible caspase 9 (iC9) was employed. This construct, calledD^(FS)iC9, selects for T cells expressing D^(FS) iC9 in the presence ofMTX and ablates D^(FS)iC9⁺ T cells in the presence of drug thatactivates iC9 to induce apoptosis. Based on the above findings, theDHFR^(FS) in D^(FS)iC9 could be used to modulate and potentially ablatethe expression of a transgene of interest which is otherwise too toxicto express without regulation. Interleukin-12 (IL-12) is such atransgene. IL-12 is a cytokine capable of inducing a strong immuneresponse against tumor from tumor specific T cells. However, systemicIL-12 is highly toxic and of low efficacy. Presented here is analternative approach where IL-12 is expressed cis to TYMS^(SS) in orderto decrease and stabilize the expression level of IL-12. In FIG. 5K, aflow plot demonstrates the expression of IL-12 cis expressed withTYMS^(SS) and iC9 cis expressed with DHFR^(FS) expression. The donorcells were either left untreated or treated with high doses of MTX for 7days. This expression pattern appears to indicate that IL-12 can bestably expressed even in prolonged toxic doses of MTX. A furtheranalysis of similarly manipulated donors (FIG. 5L) demonstrates thepotential of TYMS^(SS) when co-expressed with DHFR^(FS) to stabilize theexpression of the potentially toxic transgenes of interest—IL-12.

T cells from the experiment shown in FIG. 3D were also subjected tovarying concentrations of MTX. On day 35, T cells received anti-CD3/CD28stimulation and were subjected to a range of MTX from 0 to 1 μM for 72hours. On day 35, no T cell group significantly expressed DHFR^(FS), asindicated by co-expressed eGFP, above background (FIG. 3D-I). However,DHFR^(FS)+ T cells selected with MTX alone persisted enough tosignificantly improve survival when MTX was re-introduced atconcentrations up to 0.5 μM MTX (FIG. 7B). Flow plots in FIG. 7Ademonstrate MTX-dependent increases in transgene expression and improvedsurvival for transgene expressing T cells for one donor. It should benoted that the addition of TYMS^(SS) in [DHFR^(FS+) & TYMS^(SS)]⁺ Tcells permitted the survival of transgene negative cells at 1 μM MTX,which was not seen in TYMS^(SS neg) T cells subjected to MTX (FIG. 7C).

D. AThyR Permits Independent Selection for Transgenes of Interest

AThyRs are human proteins and therefore have lower immunogenicity inhumans than NeoR or similar drug resistance transgenes, typicallyoriginating from bacteria. Thus, using AThyRs to select transgenes ofinterest is desirable due to lower immunogenicity, and ease of use invitro. As a demonstration, the suicide gene inducible caspase 9 (iC9)was selected by co-expressing iC9 with DHFR^(FS) in a constructdesignated D^(FS) iC9 (FIG. 8A). Current methods to select iC9 utilizesurface-expressed antigen and isolation by magnetic beads. However, thismethod of selection is more labor intensive than adding drug and doesnot add the functionality of AThy resistance. The D^(FS)iC9 plasmidsignificantly selected for survival in T cells after 7 days of AaPCbased stimulation including days 2-7 days in 0.1 μM MTX (FIG. 8B). Next,D^(FS)iC9 was co-electroporated with CAR to express in T cells. The CARwas specifically selected by a CAR exodomain binding ligand (CARL)⁺ K562AaPC (Rushworth et al., supra) while D^(FS)iC9 was selected using 0.1 μMMTX. After days 2-14 in 0.1 μM MTX, CAR+D^(FS)iC9+ T cells were restedfrom MTX or selected for another 7 days in 0.1 μM MTX. T cells selectedin 0.1 μM MTX from day 2-21 are shown in FIG. 8C compared tomock-electroporated T cells. As before, there is no selection towardsCD4⁺ T cell predominance following MTX selection by day 21.

These cells also demonstrated cytotoxicity at the levels expected forthe given 5:1 target to effector ratio (FIG. 8D). Co-expressingDHFR^(FS) with iC9 rather than CAR added the potential to ablate T cellsthrough the addition of iC9 chemical inducer of dimerization AP20187(FIG. 8E). The addition of AP20187 significantly depleted resting CAR⁺ Tcells independent of MTX. This demonstrates that D^(FS)iC9 caneffectively select for iC9 expression and deplete genetically-modified Tcells as necessary. The use of DHFR^(FS) has the advantage of selectingtransgene expression in T cells independent of antigen-specificity andantigen expression.

Example 2 Materials and Methods

Healthy donor derived peripheral blood from MDACC Blood Bank, Houston,Tex., was subjected to density gradient centrifugation to isolatemononuclear cells which were either rested in complete media (CM) orfrozen as previously outlined. The use of rested or frozen peripheralblood derived mononuclear cells (PBMC) is outlined in each experiment. Tcells from PBMC were stimulated using thawed OKT3 antibody-loaded K562clone #4, an activating and propagating cell (AaPC). See Singh H, et al,PloS one 2013, 8(5). The presence of mycoplasma was tested in AaPCbefore stimulation of T cells. Cell counting was accomplished by 0.1%Trypan Blue (Sigma-Aldrich, T8154) exclusion using automated cellcounting (Nexcelcom, Lawrence, Mass.). Cell Isolation was accomplishedusing magnetic bead based sorting with the CD4+, CD25+ Regulatory T CellIsolation Kit following the manufacturer's instructions (MiltenyiBiotec, San Diego, Calif., 130-091-301). Briefly, CD4⁺ T cells werenegatively selected before sorting one time with anti-CD25 beads wasused to differentiate between effector T cells (CD25^(neg)) and T^(reg)(CD25^(pos)).

Culture Conditions: Acellular stimulation was accomplished as previouslydescribed using soluble anti-CD3—30 ng/mL, anti-CD28—100 ng/mL, andhuman IL-2—50 IU/mL, as previously described. When indicated, thefollowing drugs were used: 5-FU, MTX, cisplatin (CDDP), pemetrexed,raltitrexed, G418, hygromycin B, zeocin, rapamycin, metformin,AICARtf/inosine monophosphate (IMP) cyclohydrolase (ATIC) dimerizationinhibitor (iATIC) (Table 5). Acellular stimulation experiments receivedaddition of toxic drug or treatment on the same day as stimulation.

TABLE 5 Chemical Agents Agent Manufacturer ID No. 5-fluorouracil APPPharmaceuticals, Schaumburg, IL NDC 63323-117-10 Methotrexate Hospira,Lake Forest, IL NDC 61703-350-38 CDDP Pfizer, New York, NY NDC0069-0084-07 Pemetrexed Lilly, Indianapolis, IN NDC 0002-7640-01Raltitrexed Abcam Biochemicals, Cambridge, MA Ab142974 iATIC EMDMillipore 118490 G418 Invivogen, San Diego, CA Ant-gn-1 HygromycinInvivogen Ant-hg-1 Zeocin Invivogen Anti-zn-1 Rapamycin Wyeth,Philadelphia, PA NDC 0008-1030-04

DNA Expression Plasmids:

Selection vectors: FLAG-DHFR^(FS)-2A-eGFP pSBSO (noted as DHFR^(FS)-GFP(DG)), FLAG-TYMS^(SS)-2A-eGFP pSBSO (noted as TYMS^(SS)-GFP (TSG)),NLS-mCherry pSBSO (RFP), FLAG-TYMS^(SS)-2A-NLS-mCherry pSBSO (noted asTYMS^(SS)-RFP (TRG)), Neomycin Resistance (NeoR)-2A-eGFP pSBSO (noted asNeoR-GFP (NRG)), and Myc-ffLuc-NeoR pSBSO (NRF), were designedconstructed and utilized as previously described. Sleeping Beauty (SB)indirect/direct repeat (IR/DR) sites were present in each construct toinduce genomic integration with SB transposase. Each transgene wasexpressed by elongation factor 1 alpha (EF1α) promoter.

Genetic Transformation and Propagation of Cells:

The Amaxa Nucleofector® II was utilized to transform human PBMC, where1-2*10⁷ thawed PBMC were electroporated in Amaxa T cell Nucleofectorsolution using program U14, as previously described. The next day, PBMCwere stimulated with CM with AaPC at a ratio of 1:1 including 50 IU/mLIL-2. The co-culture of T cells and AaPC was maintained at 1*10⁶cells/mL with each subsequent stimulation. Outgrowth of T cells waspromoted by re-stimulated of co-cultures every 7 days with IL-2 and AaPCat the concentrations noted. Fresh IL-2 was added when media was changedbetween stimulations. During transgenic experiments, drugs were added 48hours after co-culture initiation and maintained at the givenconcentration until day 14. After day 14, no drugs were added to T cellcultures.

Western Blot:

When noted, T cells were removed from cultures for western blot bycentrifugation of 1*10⁶ T cells, and rapid freezing of the cell pelletin liquid nitrogen. T cell pellets were lysed and prepared with 50 mMTris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate, 1 mMphenylmethylsulfonyl fluoride, 150 mM p-nitrophenyl phosphate and 0.3 μMAprotinin, pH 7.4. SDS-PAGE separated proteins and primary antibodiesnoted in Table 6 were used to detect the presence of protein viachemiluminescence.

TABLE 6 Western Blot Antibodies Antibody Manufacturer Cat. No. DilutionAMPKα Cell Signaling Technology (CST), 2603S 1:1000 Danvers, MA p-AMPKαCST 2535S 1:1000 (T172) S6 CST 2317S 1:1000 p-S6 CST 3945S 1:1500(S235/236) Actin Sigma A2228 1:10000 Hsp-70 Santa Cruz Biotechnology,Dallas, SC-24 1:5000 TX eEF2 LifeSpan Biosciences, Seattle, WA LS-B8940p-eEF2 (T56) LifeSpan Biosciences LS-C198899

Flow Cytometry:

Cultured T cells were washed in FACS staining solution[95] beforesurface antibody staining was performed in FACS staining solution withfluorochrome-conjugated antibodies at 4° C. for at least 30 minutes.Intracellular transcription factor and cytokine staining utilized theFoxP3/transcription factor staining buffer set manufacturer's protocol(eBioscience, 00-5523-00), and was performed following surface staining.The BD FACSCalibur (BD Biosciences) analyzed most samples expressingFoxP3. Antibody targets, concentrations, and manufacturers are listed inTable 7. Flow cytometry data analysis utilized FlowJo v 10.0.5 (TreeStar Inc., Ashland, Oreg.). Flow cytometric imaging of cells stained forphosphorylated antigens was accomplished using the ImageStreamX Mark II(Amnis, Seattle, Wash.) with the following protocol; after surfacestaining, samples were fixed in 100% methanol (Sigma) for 1 hour at 4°C. before washing and staining in FoxP3/transcription factor stainingbuffer set wash buffer as outlined by the manufacturer's protocol.Analysis of image cytometry data utilized Amnis IDEAS v 6.0.

TABLE 7 Flow Cytometry Antibodies Antibody Manufacturer Cat. No.Dilution CD3-APC BD Pharmingen 340661 1:33 CD3-PerCP-Cy5.5 BD Pharmingen340949 1:33 CD4 FITC BD Pharmingen 340133 1:33 CD4-PE BD Pharmingen347327 1:33 CD4-PerCP-Cy5.5 BD Pharmingen 341645 1:33 CD8-APC BDPharmingen 340659 1:33 CD25-APC BD Pharmingen 555434 1:33 CD39-APC BDPharmingen 560239 1:33 CD45RO-APC BD Pharmingen 559865 1:33 CD152-APC BDPharmingen 555855 1:33 KI-67-AF647 BD Pharmingen 561126 1:50 Annexin VBD Pharmingen 556422 1:20 7-AAD BD Pharmingen 559925 1:20 PropidiumIodide BD Pharmingen 556463 FoxP3-PE eBiosciences 12-4777-42 1:20Helios-APC Biolegend 137222 1:05 LAP-APC Biolegend 349608 1:20 IFN-g-APCBiolegend 502516 1:20 IL-2-APC Biolegend 500315 1:20 p-eEF2 (T56)LifeSpan LS-C198899 1:20 Biosciences p-AMPKα (T172) AbCam Ab133448 1:20CD4-Pacific Blue BD Pharmingen 558116 1:33 p-S6 (S244) - BD Pharmingen560465 1:20 AF647 Goat anti-Rabbit - Life Technologies A-11034 1:100AF488

Thymidine Incorporation Assay:

A thymidine incorporation assay was performed with anti-CD3/CD28 andIL-2 used to stimulate each well containing 2*10⁵ viable cells. Varyingratios of effector T cells (T_(eff)) to T_(reg) were combined in eachwell and all wells were run in triplicate in U-bottom 96 well plates. At48 hours 1 μCi [³H] Thymidine (Perkin-Elmer, Waltham, Mass.) was addedto each well, and 24 hours later the cells were assessed forradioactivity on a Top Count NXT (Perkin-Elmer). T_(reg) mediatedsuppression of growth was determined by the following equation: (NoTreatment T_(eff) [cpm]−(T_(reg) & No Treatment T_(eff) [cpm]))/NoTreatment T_(eff) [cpm].

Statistical Analysis:

Graphical representation and statistical analysis of data was performedwith Prism v6.0 (Graph Pad Software Inc., La Jolla, Ca). One-Way ANOVAwas used when appropriate with Tukey's or Dunnett's multiple comparisontests as applicable, non-Gaussian distributions were assessed by theKruskall-Wallis test followed by Dunn's multiple comparison test. Totalcell counts and expression data involving T_(CD4, FoxP3) tended to benon-Gaussian in distribution. Single variable tests (experimental vs.control) were made using the Mann-Whitney test. Statistical significancewas designated as α<0.05.

Results

Drug selection of TCD4, FoxP3 by MTX occurs in part through toxicity. Inorder to determine how MTX contributes to the selection ofT_(CD4, FoxP3), freshly derived PBMC were stimulated with anti-CD3/CD28antibodies and IL-2 in the presence of cytotoxic drugs or lethalγ-irradiation. After 7 days there was a significant difference insurvival markers Annexin V and 7-AAD in stimulated T cells receiving anycytotoxic insult with stimulation (FIG. 1B-I). The selection ofT_(CD4, FoxP3) was not as consistent as cytotoxicity. Following 7 daysof stimulation, 2 Grey γ-irradiation significantly increased the amountof T_(CD4, FoxP3) in the surviving population (FIG. 1B-II). This lethaltreatment did not target a common pathway being considered, nor didcisplatin, yet both increased T_(CD4, FoxP3). However, theT_(CD4, FoxP3) increase induced by cisplatin is insignificant.Significant increases were derived from 5-FU and MTX. With the exceptionof ribosomal elongation inhibitor G418, each cytotoxic treatmentappeared to increase the percentage of surviving T_(CD4, FoxP3). SeeBar-Nun S. et al., Biochimica et biophysica acta 1983, 741(1):123-127.This pattern of increasing T_(CD4, FoxP3) percentage in the face ofvaried cytotoxic insult suggests a common pathway that can be enhancedby certain drugs. Without wishing to be bound by theory, this pathway islikely related to the reduced proliferation rate of T_(reg), and appearsto be ribosomally mediated as G418 can inhibit this general trend ofincreasing T_(CD4, FoxP3) percentage. See Cao M. et al., Internationaljournal of radiation biology 2011, 87(1):71-80.

The findings of T_(reg) depletion with G418 and T_(reg) selection by MTXwere further evaluated for dose dependence by stimulating thawed PBMCwith anti-CD3/CD28+IL-2 for 7 days, as before. G418 was significantlycytotoxic at all doses tested, but significantly depleted T_(CD4, FoxP3)at two moderate drug doses (FIG. 10C). MTX was also cytotoxic at alldoses tested, but had significant elevation of T_(CD4, FoxP3) at lowerdoses (FIG. 10D). Rapamycin (Rapa) was used as a T_(reg) selectioncontrol_([138]) and showed similar T_(CD4, FoxP3) selection at amoderate drug concentration independent of cytotoxicity, which onlyoccurred at the highest doses (FIG. 10F). The selection for or againstT_(reg) at moderate drug doses rather than higher doses suggests thatT_(reg) have a narrow therapeutic window for drug induced selection ordepletion. A specific inhibitor of ATIC_([142]) was used to test whetherMTX mediates selection of T_(CD4, FoxP3) through inhibition of ATIC.Without wishing to be bound by theory, inhibition of AICARtf or theheterodimeric complex ATIC, in which AICARtf is found, increases AICAR.FIG. 10E demonstrates that ATIC inhibition alone was neither cytotoxicnor selective for T_(CD4, FoxP3). Further analysis of flow plotsrepresented by the same donor in FIG. 10G show expression of CD4 andFoxP3 for several of the drugs used. Use of iATIC characteristicallymediated increased expression of FoxP3 in CD4+ T cells similar to thatof Rapa, but did not inhibit proliferation of FoxP3_(neg) T cells asMTX, G418, or Rapa. Thus, iATIC enhanced FoxP3 expression in CD4+ Tcells but diluted these cells by permitting proliferation of FoxP3_(neg)T cells. It appears that MTX mediated selection of T_(CD4, FoxP3) occursby depletion of rapidly proliferating effector T cells and enhancementof FoxP3 expression via a pathway similar to Rapa that includesribosomal inhibition. The increased susceptibility of T_(regs) toribosomal inhibitor G418 solidifies this relationship between enhancedFoxP3 expression and increased susceptibility to ribosomal inhibition.

T_(regs) are preferentially expanded in primary T cells resistant to theanti-folate and anti-thymidine actions of MTX. It was hypothesized thatregulatory T cells were inhibiting CD8+ T cells proliferation followingdrug selection. To test this hypothesis, drug resistant T cells werederived by transformation with DHFR^(FS), TYMS^(SS), NeoR, or acombination, and numerically expanded as previously described. Briefly,transformed T cells were selected in the presence of 0.1 μM MTX, 5 μM5-FU, or 1.6 mM G418 as designated from day 2 to 14 while stimulationwith OKT3-loaded AaPC and 50 IU/mL IL-2 occurred every 7 days until day35. See Singh H. et al., PloS one 2013, 8(5). Initial testing forT_(regs) by elevated expression of FoxP3 in the CD4⁺ T cell populationdemonstrated there was a significant T_(CD4, FoxP3) percentage increasein DHFR^(FS) expressing T cells. Selection using MTX in comparison tomock-electroporated (No DNA) T cells on Day 21 showed this increase(FIG. 11), and this increase persisted to Day 35 when 5-FU was combinedwith MTX during selection (FIG. 12A). The transgenic T cells were almostentirely CD4⁺ in each experimental population after selection, but thepredominance of T_(regs) appeared to often exceed the 5-10% typicallyfound in the un-manipulated CD4⁺ T cell compartment. Markers of T_(reg)function were also assessed. Low IL-2 expression_([ ]) is a known traitof T_(regs) and is assessed with FoxP3 expression. The percentage of theT cell population with a FoxP3_(pos), IL-2_(neg) expression pattern isshown in FIG. 12B. Expression of latency associated peptide (LAP)—a partof the TGF-β complex and strongly associated with activated T_(reg), andis seen in FIG. 12C.

The transgenes DHFR^(FS) and TYMS^(SS) were compared individually and incombination to the control selection vector NeoR and un-treated No DNA Tcells. Selection towards T_(reg) in this experiment may be noted in FIG.12A, B, C-I. This experiment demonstrated that [DHFR^(FS)-GFP (DG) &TYMS^(SS)-RFP (TSR)]⁺ T cells selected in MTX+5-FU had an increasedpopulation of cells characteristic of T_(reg) when compared tomock-transformed T cells. To further elucidate the contribution ofDHFR^(FS) and TYMS^(SS) to T_(reg) selection, NeoR was co-electroporatedwith DHFR^(FS), TYMS^(SS), or the combination. The addition of NeoRpermitted equivalent selection of DHFR^(FS), TYMS^(SS), and thecombination in all T cell populations. With un-transformed T cellsremoved, it became clear that DHFR^(FS) alone, but not TYMS^(SS) alonecould select for cells characteristic of T_(regs) (FIGS. 12A, B, andC-II). [DG & TSR]⁺ T cells continued to select for cells with T_(reg)features. Finally, the contribution of TYMS^(SS) to the selection ofT_(reg) by DHFR^(FS) was assessed by co-electroporation of TSR or acontrol vector—RFP. The characteristics of T_(regs) from this experimentare shown in FIGS. 12A, B, and C-III. This experiment demonstrates thatselection of DHFR^(FS) with MTX can enhance outgrowth of T_(reg) andthat 5-FU enhances this selection independent of TYMS^(SS). Selection ofT_(reg) benefits from folate rescue by DHFR^(FS). This is expected asfolate is known to play a role in T_(reg) survival. See Kunisawa J. etal., PloS one 2012, 7(2):e32094. Surprisingly, selection of T_(reg) didnot require de novo thymidine synthesis as TYMS^(SS), which alleviatesMTX and 5-FU inhibition of TYMS, was dispensable.

Previous findings showed survival and toxicity of 5-FU in PBMC ismediated by TYMS and an alternative mechanism. See Eisenthal A et al.,Anticancer research 2009, 29(10):3925-3930. Combining the knownmechanisms of T_(reg) selecting drugs MTX, 5-FU, and rapamycin yieldedthe diagram in FIG. 13, which details how each drug interacts withribosomal function. It was noted in an experiment depicted inSupplemental FIG. 1A that Neomycin resistance gene rescuedT_(CD4, FoxP3) from the treatment of G418. This finding suggests that aspecific action of G418 is responsible for T_(CD4, FoxP3) depletion, andthis phenomenon was further explored.

Ribosomal Inhibition by aminoglycoside G418 selectively depletesreplicating T_(CD4, FoxP3). Thawed PBMC were activated withanti-CD3/CD28+IL-2 for 7 days in the presence of alternative doses ofG418, Hygromycin B—a different aminoglycoside, Zeocin—a DNA targetingantibiotic, and Rapa to assess the dose dependent selection or depletionof T_(CD4, FoxP3) by aminoglycosides (FIG. 14A). Depletion ofT_(CD4, FoxP3) is again noted in the presence of aminoglycoside G418.The alternative aminoglycoside—hygromycin—developed an insignificantincrease in T_(CD4, FoxP3) at 0.2 mM hygromycin. This increasesignificantly decreased with higher doses of hygromycin—1.5 and 2.3 mM.Hygromycin showed no significant depletion of T_(CD4, FoxP3) fromuntreated control.

Ribosomal Inhibition by aminoglycoside G418 selectively depletesreplicating T_(CD4, FoxP3). Thawed PBMC were activated withanti-CD3/CD28+IL-2 for 7 days in the presence of alternative doses ofG418, Hygromycin B—a different aminoglycoside,_([146]) Zeocin—a DNAtargeting antibiotic, and Rapa to assess the dose dependent selection ordepletion of T_(CD4, FoxP3) by aminoglycosides (FIG. 14A). Depletion ofT_(CD4, FoxP3) is again noted in the presence of aminoglycoside G418.The alternative aminoglycoside—hygromycin—developed an insignificantincrease in T_(CD4, FoxP3) at 0.2 mM hygromycin. This increasesignificantly decreased with higher doses of hygromycin—1.5 and 2.3 mM.Hygromycin showed no significant depletion of T_(CD4, FoxP3) fromuntreated control.

This dose dependent depletion of T_(CD4, FoxP3) is consistent with thatseen for G418, and was not noted with increasing doses Zeocin or Rapa.An increase of T_(CD4, FoxP3) was noted with increasing doses of Zeocin,yet this was insignificant, similar to that seen for other cytotoxicdrugs in FIG. 10B-II. A representative flow plot of CD4 and FoxP3expression from the same donor can be seen in FIG. 14B. Here, the trendscan be visualized.

It was considered that polyclonal stimulation may play some part in theG418 depletion of T_(CD4, FoxP3). To test this, PBMC were rested in CMfor 9 days after thawing +/−G418 and tested for the presence ofT_(CD4, FoxP3). Significant depletion of T_(CD4, FoxP3) by G418persisted under resting conditions (FIG. 14C—left panel). This wasreplication dependent as CD4+,FoxP3+,Ki-67+ cells showed significantG418 mediated depletion while CD4+,FoxP3+,Ki-67neg cells were notsignificantly depleted by the same post-Hoc measure (FIG. 14C—rightpanel). Representative flow diagrams of resting PBMC in FIG. 14D—upperpanel show the loss in expression of FoxP3 for CD4+ T cells aftertreatment with G418. An alternative view of Ki-67 and FoxP3 expressionin FIG. 14D—lower panel demonstrates that FoxP3neg T cells continue toproliferate in the presence of G418, further supporting the selectivetargeting of G418 to T_(CD4, FoxP3) at this concentration. Thus,proliferating T_(CD4, FoxP3) are depleted following treatment withaminoglycoside G418.

As G418 and hygromycin are considered toxic to live animals, gentamicin,an aminoglycoside well known for its use in humans and animal models,was tested for selective TCD4, FoxP3 depletion. See Lopez-Novoa J M. etal., Kidney international 2011, 79(1):33-45. FIG. 3E depicts thisdepletion of T_(CD4, FoxP3) in resting PBMC after 7 days anddemonstrates the consistent action of aminoglycosides in depleting TCD4,FoxP3. It was next tested whether depletion of T_(CD4, FoxP3)corresponded with a loss of T_(reg) marker expression or selectiveT_(reg) toxicity.

Sorted Treg differentiate the effects of MTX, 5-FU, and G418 onselection in bulk PBMC. Magnetic sorting for CD4 and CD25 expressingPBMC yielded a CD4+CD25+ population that is widely considered to containT_(reg), and a CD25_(neg) population of effector T cells (T_(eff)). SeeMiyara M. et al., Immunity 2009, 30(6):899-911. These populations weretreated with the same concentrations of MTX, 5-FU, G418, or notreatment, as above, for the first 7 days of co-culture with AaPC. Afterthis period of time, co-culture continued without drug by stimulatingwith AaPC every 7 days until Day 21. Cells were assayed at this time forexpression of CD25, CTLA-4, LAP, and IL-2, as before. The experimentaloutline can be seen in FIG. 15A. A [³H] thymidine incorporation assaywas also performed to determine the effect of each drug on thefunctionality of propagated T_(reg).

When the surviving CD4⁺ cells were assayed on day 21 it was found thatno drug significantly selected for T_(CD4, FoxP3) in the T_(eff)compartment, nor did MTX and 5-FU improve selection for T_(CD4, FoxP3)in the T_(reg) compartment (FIG. 15B). The most consistent finding wasthat G418 persistently decreased surviving T_(reg) following drugtreatment. This was demonstrated by loss of surviving T_(CD4, FoxP3)(FIG. 15B). T_(reg) markers such as CD25 (FIG. 15C-I), CTLA-4 (FIG.15C-II), decreased IL-2 expression (FIG. 4C-III), or LAP (FIG. 15C-IV),in combination with FoxP3 expression was also decreased followingstimulation on day 21. Thus, T_(reg) are lost, likely due to toxicity ofG418, rather than inhibited as 2 weeks of growth promoting co-cultureconditions could not sufficiently restore T_(regs) following G418treatment.

The T_(reg) promoting properties of MTX and 5-FU appeared to depend inpart upon the presence of T_(eff), as the enhanced selection ofT_(CD4, FoxP3) was no longer noticeable after T_(eff) were removed fromthe culture system (FIG. 15B). The improved selection towards T_(reg)phenotypes may have been accomplished by depletion of T_(eff) which areknown to contaminate T_(reg) sorting._([113]) It is likely that theability of T_(reg) to survive the cytotoxic insult of MTX or 5-FU incomparison to T_(eff) was a primary component of the enhanced selection.Although there was a trend towards improved selection of T_(reg)phenotypes (FIG. 15C-I, II, III) when MTX or 5-FU was used, there was nosignificant difference for expression of CD25, CTLA-4, or loss of IL-2.However, the T_(reg)-specific marker LAP was significantly increased byearly treatment with MTX or 5-FU (FIG. 15C-IV). As LAP was the onlyincreased marker of those assayed, it is likely that LAP and theassociated expression of TGF-β_([143]) was the probable cause forimproved suppression of MTX and 5-FU treated T_(reg) above untreatedT_(reg) (FIG. 15D). Thus, MTX and 5-FU appear to have two components inenhancing selection of T_(reg): 1) T_(eff) are selectively depleted byMTX and 5-FU, and 2) MTX and 5-FU increase the expression of LAP weeksafter treatment.

Stimulation of T_(CD4, FoxP3) enhances AMPK activation and leads toinhibition of eEF2—a factor that plays a role in translationalelongation. AMPK is hypothesized to play a role in selection ofT_(CD4, FoxP3), as noted above (FIG. 13). Furthermore, enhancedactivation of AMPK may lead to inhibition of eEF2 in T_(CD4, FoxP3). SeeBrowne G J. et al., The Journal of biological chemistry 2004,279(13):12220-12231. Preferential inhibition of translational elongationcould explain selection for T_(CD4, FoxP3) in the presence of manycytotoxic drugs and depletion of T_(CD4, FoxP3) in the presence ofinhibitors of translational elongation. This was tested by assessingphosphorylation of AMPK 24 hours after activation of PBMC using flowcytometry (FIGS. 16A & B) and imaging cytometry (FIG. 16C). Thephosphorylation of AMPK on T172 indicates activation and was enhanced instimulated over unstimulated T_(CD4, FoxP3). See Hardie D G et al.,Diabetes 2013, 62(7):2164-2172. This enhanced activation of AMPK wasincreased in CD4⁺, FoxP3_(neg) T cells (FIG. 16A—upper panel) as well,but the significant increase (p=0.03 by t-test) did not persistfollowing post-hoc analysis. Likewise, flow plots of activated AMPK withFoxP3 show this enhancement of AMPK activation is much more noticeablein the FoxP3-expressing subset (FIG. 16B—upper panel). See MacIver N Jet al., Journal of immunology 2011, 187(8):4187-4198. A marker oftranslational initiation—S6—is susceptible to mTOR regulation, and isphosphorylated when active. See Mahoney S J et al., Progress inmolecular biology and translational science 2009, 90:53-107.Phosphorylation of S6 (p-S6) was significantly enhanced inT_(CD4, FoxP3) following stimulation (FIG. 16A—lower panel), which waspreviously shown by Cabone et al. See Carbone F. et al., Nature medicine2014, 20(1):69-74. While p-S6 increased in the FoxP3_(neg) T cells(p=0.01 by t-test), this increase was not significant following post-hocanalysis. The enhancement of p-S6 is observable in the representativeflow plot for FIG. 16B—lower panel. The activation of metabolicregulators AMPK and S6 was enhanced in both FoxP3⁺ and FoxP3^(neg) CD4⁺T cells following activation, but the increase was only significant inT_(CD4, FoxP3) in a Two-Way ANOVA with post-hoc Sidak's test. Theincreased activation of AMPK and S6 following activation ofT_(CD4, FoxP3) can be seen with image cytometry profiles shown in FIG.16D before—top panel—and after stimulation with anti-CD3/CD28 andIL-2—bottom panel. The same compensation and visualization were appliedto each panel making the top and bottom panels comparable.

Without wishing to be bound by theory, enhanced activation of AMPK inT_(CD4, FoxP3) suggests translational elongation may be inhibited byphosphorylation of eEF2 and could account for the increased survival ofT_(CD4, FoxP3) in the presence of cytotoxic drugs and susceptibility toinhibitors of translational elongation, like aminoglycosides. The sameexperiment as in FIG. 16 A-C was performed to assess the inactivation ofeEF2 by phosphorylation at T56.[135] Image cytometry was used toquantify and visualize all events. FIG. 16D demonstrates a significantincrease in phosphorylation of eEF2 in the same subset of Tcells—T_(CD4, FoxP3)—following stimulation. Also, inhibitoryphosphorylation of eEF2 was significantly increased above stimulatedFoxP3_(neg) T cells, which was not noted with AMPK or S6phosphorylation. The increased phosphorylation of eEF2 only instimulated T_(CD4, FoxP3) suggests that T_(CD4, FoxP3) would havedecreased replicative capacity upon stimulation, as shown by Cao et al.Decreased levels of active eEF2, which inhibit progression through thecell cycle, suggest that increased phosphorylation of eEF2 may accountfor the survival of T_(CD4, FoxP3) in cytotoxic environments, which wasnoted in FIG. 10. Similarly, decreased translational capacity would makeT_(CD4, FoxP3) increasingly susceptible to inhibitors of translationalelongation, as was shown with aminoglycosides in FIG. 14. Therefore, theactivity of eEF2 may be the primary factor influencing both selectionand depletion of T_(reg) in these studies.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

All cited patents and publications referred to in this application areherein incorporated by reference in their entirety.

1-74. (canceled)
 75. An isolated engineered mammalian T cell expressinga first transgene and TYMS^(SS), wherein said T cell comprises (1) anucleotide sequence encoding the first transgene and (2) a nucleotidesequence encoding TYMS^(SS).
 76. The isolated engineered mammalian Tcell of claim 75, wherein the nucleotide sequence encoding the firsttransgene and the nucleotide sequence encoding TYMS^(SS) are operablylinked.
 77. The isolated engineered mammalian T cell of claim 75,wherein the nucleotide sequence encoding the first transgene and thenucleotide sequence encoding TYMS^(SS), upon expression, are encoded onthe same mRNA.
 78. The isolated engineered mammalian T cell of claim 75,wherein the nucleotide sequence encoding the first transgene and thenucleotide sequence encoding TYMS^(SS) are separated by an internalribosomal entry site (IRES) or a ribosomal slip sequence.
 79. Theisolated engineered mammalian T cell of claim 75, wherein nucleotidesequence encoding the first transgene is positioned 3′ relative to thenucleotide sequence encoding TYMS^(SS).
 80. The isolated engineeredmammalian T cell of claim 75, wherein the first transgene is a chimericantigen receptor (CAR) construct, a polypeptide hormone, a suicide gene,a T-cell receptor (TCR), a growth factor, or a cytokine.
 81. Theisolated engineered mammalian T cell of claim 80, wherein the cytokineis IL-12 or IL-15.
 82. The isolated engineered mammalian T cell of claim75, further comprising (3) a nucleotide sequence encoding DHFR^(FS). 83.The isolated engineered mammalian T cell of claim 82, wherein thenucleotide sequence encoding DHFR^(FS) is operably linked to anucleotide sequence encoding a second transgene.
 84. The isolatedengineered mammalian T cell of claim 83, wherein the nucleotide sequenceencoding the second transgene and the nucleotide sequence encodingDHFR^(FS), upon expression, are encoded on the same mRNA.
 85. Theisolated engineered mammalian T cell of claim 83, wherein the nucleotidesequence encoding the second transgene and the nucleotide sequenceencoding DHFR^(FS) are separated by an internal ribosomal entry site(IRES) or a ribosomal slip sequence.
 86. The isolated engineeredmammalian T cell of claim 83, wherein the second transgene is a suicidegene, CAR, TCR, polypeptide hormone, cytokine, chemokine, ortranscription factor.
 87. The isolated engineered mammalian T cell ofclaim 75, wherein the isolated engineered mammalian T cell is a T helpercell (TH cell), cytotoxic T cell (Tc cell or CTL), memory T cell (TCMcell), effector T cell (TEM cell), regulatory T cell (Treg cell; alsoknown as suppressor T cell), natural killer T cell (NKT cell), mucosalassociated invariant T cell, alpha-beta T cell (Tαβ cell), orgamma-delta T cell (Tγδ cell).
 88. A method of treating a patient with acancer comprising to administering to the patient a therapeuticallyeffective amount of the isolated engineered mammalian T cells of claim75.
 89. A method of enriching for regulatory T cells in a population ofT cells isolated from a mammal, the method comprising contacting thepopulation of T cells with a thymidine synthesis inhibitor selected fromthe group consisting of methotrexate (MTX), 5-FU, Raltitrexed, andPemetrexed, or a combination thereof, to selectively deplete effector Tcells in the population.
 90. The method of claim 89, wherein thepopulation of T cells isolated from a mammal is contacted with both MTXand 5-FU.
 91. The method of claim 89, wherein the T cells express one orboth of DHFR^(FS) and TYMS^(SS).
 92. A method for selecting a T cellexpressing a transgene of interest comprising: a) applying a thymidinesynthesis inhibitor to a plurality of T cells that comprises a T cellexpressing the transgene of interest and TYMS^(SS); and b) selecting forone or more T cells surviving after seven or more days of application ofthe thymidine synthesis inhibitor, wherein the one or more surviving Tcell(s) expresses the transgene of interest and TYMS^(SS).
 93. Themethod of claim 92, wherein the T cell expressing the transgene ofinterest and TYMS^(SS) further expresses DHFR^(FS).
 94. The method ofclaim 92, wherein the thymidine synthesis inhibitor is selected from thegroup consisting of methotrexate (MTX), 5-FU, Raltitrexed, andPemetrexed.